ES2997833T3 - Formulations of human anti-rankl antibodies, and methods of using the same - Google Patents

Abstract

Described herein are aqueous pharmaceutical formulations comprising denosumab or another human anti-RANKL monoclonal antibody or a portion thereof, along with pH characteristics, buffer systems, and amino acid aggregation inhibitors. Also described are the formulation presentations for use, e.g., in a single-use vial, a single-use syringe, or a glass container, methods of using the formulations, and articles for preventing or treating diseases and related kits. (Automatic translation with Google Translate, no legal value)

Description

DESCRIPTION

Human anti-RANKL antibody formulations, and methods of using the same

BACKGROUND

Field of dissemination

The invention relates to human anti-RANKL monoclonal antibodies, including high concentration aqueous formulations of denosumab and biosimilars thereof.

Brief description of the related technology

Denosumab is commercially available in dissolution forms in concentrations of 60 mg/ml and 70 mg/ml.

Increasing concentrations of protein formulations can cause stability issues, for example, aggregation resulting in the formation of high-molecular-weight species (HMWS). HMWS, particularly those that retain most of the native configuration of the monomeric counterpart, can be of particular concern in some protein formulations. Aggregation can also potentially affect the subcutaneous bioavailability and pharmacokinetics of a therapeutic protein.

Fill-finish operations, as well as dispensing, may involve circulating protein solutions through piston pumps, peristaltic pumps, or injection needles. These processes can impart shear and mechanical stress, which can cause protein denaturation and result in aggregation. This phenomenon can be exacerbated as protein solutions become more concentrated.

US patent document 2011/0060290 relates to stabilized compositions of RANKL-specific binding agents, TNF-specific binding agents, and/or IL-1R1-specific binding agents in vessels.

SUMMARY

According to the present invention, there is provided a first-time disclosure demonstrating that the addition of an amino acid aggregation inhibitor to an aqueous solution comprising a high concentration of an anti-RANKL antibody leads to a reduced amount of antibody aggregates formed over time, as well as a slower rate of formation of such aggregates. The present disclosure also provides an effect of pH on aggregate formation in concentrated aqueous solutions of anti-RANKL antibody, wherein decreased aggregate formation is observed when the pH of the aqueous solutions is in the range of about 5.0 to less than 5.2. The disclosure presented herein further suggested that stabilization of the anti-RANKL antibody occurs through interactions between the amino acid aggregation inhibitor and the antibody. Without wishing to be bound by any particular theory, it is contemplated that hydrophobic interactions, as well as other types of intermolecular interactions, between the amino acid aggregation inhibitor and the anti-RANKL antibody have a stabilizing effect on concentrated antibody solutions. Accordingly, the disclosure of the present invention relates to stable aqueous pharmaceutical formulations comprising a high concentration of an anti-RANKL antibody, said formulations comprising low amounts (e.g., less than about 2%) of aggregates.

In a first embodiment, the invention provides an aqueous pharmaceutical formulation comprising:

- an anti-RANKL antibody at a concentration in the range of 100 to 140 mg/ml; and

- L-phenylalanine or L-tryptophan 5 mM to 180 mM;

wherein the aqueous pharmaceutical formulation has a pH in a range of 5.0 to 5.4; and

wherein the anti-RANKL antibody comprises a heavy chain comprising SEQ ID NO: 14 and a light chain comprising SEQ ID NO: 13.

In a second embodiment, the invention provides the aqueous pharmaceutical formulation of the invention for use in (a) the treatment or prevention of a skeletal-related event (SRE) in a subject with bone metastases from solid tumors, (b) the treatment or prevention of an SRE in a subject who is a skeletally mature adult or adolescent with giant cell tumor of bone such that it is inoperable or where surgical resection is likely to result in intense morbidity, (c) the treatment of hypercalcemia of malignancy refractory to bisphosphonate therapy in a subject, (d) the treatment or prevention of an SRE in a subject with multiple myeloma or with bone metastases from a solid tumor, (e) the treatment of osteoporosis of postmenopausal women at high risk of fracture, (f) the treatment to increase bone mass in women at high risk of fracture receiving adjuvant aromatase inhibitor therapy for breast cancer, (g) the treatment to increase bone mass in men with high risk of fracture receiving androgen deprivation therapy for non-metastatic prostate cancer, (h) treatment to increase bone mass in men with osteoporosis at high risk of fracture, (i) calcium or vitamin D therapy, (j) treatment of giant cell tumor of bone in a patient in need thereof, (k) treatment of hypercalcemia of malignant tumor in a patient in need thereof, (l) treatment of osteoporosis in a patient in need thereof, or (m) increasing bone mass in a patient in need thereof.

ADDITIONAL DISCLOSURE

An aqueous pharmaceutical formulation may comprise a human anti-receptor activator of nuclear factor kappa-B ligand (anti-RANKL) monoclonal antibody or an antigen-binding portion thereof at a concentration greater than 70 mg/ml and having a pH in a range of about 5.0 to less than 5.2.

An aqueous pharmaceutical formulation may comprise a mixture of a human anti-receptor activator of nuclear factor kappa-B ligand (anti-RANKL) monoclonal antibody or an antigen-binding portion thereof and an amino acid aggregation inhibitor. The amino acid aggregation inhibitor may comprise an amino acid comprising a charged side chain, an aromatic amino acid, or a hydrophobic amino acid. The amino acid comprising a charged side chain may be an amino acid comprising a positively charged side chain, such as, for example, arginine and lysine. The aromatic amino acid may comprise a phenyl or an indole. Optionally, the aromatic amino acid further comprises a C<1>-C<6> alkyl chain between the alpha carbon and the phenyl or indole. Amino acids, including, for example, phenylalanine and tryptophan, are exemplary amino acid aggregation inhibitors. The amino acid aggregation inhibitor can be a hydrophobic amino acid that has a score greater than about 2.5 on the Kyte-Doolittle hydrophobicity scale. Optionally, the hydrophobic amino acid is valine, leucine, or isoleucine. Additional amino acid aggregation inhibitors are contemplated as described herein.

The aqueous pharmaceutical formulation may further comprise a tonicity modifier, a surfactant, a buffer, or any combination thereof.

A presentation of the formulation for storage or use is described, for example, in a single-use vial, single-use syringe, or primary glass, glass-lined, or glass-coated container. A container, optionally, a vial, prefilled syringe (PFS), or glass container, may comprise any of the aqueous pharmaceutical formulations described herein. The container may comprise about 1 mL or less (e.g., about 0.5 mL) of the aqueous pharmaceutical formulation.

Methods of preparing a stable aqueous pharmaceutical formulation comprising an anti-human receptor activator of nuclear factor kappa-B ligand (anti-RANKL) monoclonal antibody, or an antigen-binding portion thereof, may comprise combining the anti-RANKL monoclonal antibody, or antigen-binding portion thereof, at a concentration greater than 70 mg/ml with an amino acid aggregation inhibitor, a buffer, a surfactant, and optionally a tonicity modifier. Disclosed is a stable aqueous pharmaceutical formulation prepared according to any one of the methods of preparing a stable aqueous pharmaceutical formulation described herein.

Disclosed are methods of using a formulation as described herein for preventing or treating a disease responsive to a human anti-RANKL monoclonal antibody or an antigen-binding portion thereof. The use may encompass therapeutic treatment of a subject encompassing treatment or prevention of a skeletal-related event (SRE), treatment or prevention of a giant cell tumor of bone, treatment or prevention of hypercalcemia of malignancy, treatment or prevention of osteoporosis, or increasing bone mass, in a subject. For example, therapeutic treatment encompasses (a) the treatment or prevention of an SRE in a subject with bone metastases from solid tumors, (b) the treatment or prevention of an SRE in a subject who is a skeletally mature adult or adolescent with giant cell tumor of bone to the extent that it is inoperable or where surgical resection is likely to result in severe morbidity, (c) the treatment of hypercalcemia of malignancy refractory to bisphosphonate therapy in a subject, (d) the treatment or prevention of an SRE in a subject with multiple myeloma or with bone metastases from a solid tumor, (e) the treatment of osteoporosis in postmenopausal women at high risk for fracture, (f) treatment to increase bone mass in women at high risk for fracture receiving adjuvant aromatase inhibitor therapy for breast cancer, (g) treatment to increase bone mass in men at high risk for fracture receiving androgen deprivation therapy for non-metastatic prostate cancer, (h) treatment to increase bone mass in men with osteoporosis at high risk of fracture, (i) calcium or vitamin D therapy.

Disclosed are a method of preventing a skeletal-related event (SRE) in a patient in need thereof, a method of treating giant cell tumor of bone in a patient in need thereof, a method of treating hypercalcemia of malignancy in a patient in need thereof, a method of treating osteoporosis in a patient in need thereof, and a method of increasing bone mass in a patient in need thereof. The methods comprise administering to the patient an effective amount of any one of the formulations described herein. The formulation may be administered subcutaneously to the patient.

The invention provides the use of denosumab, or another human anti-RANKL monoclonal antibody or an antigen-binding portion thereof, in the manufacture of a medicament as described herein for treating a patient in need of a human anti-RANKL monoclonal antibody.

A kit may include a composition or article described herein together with a package insert, label, instructions, or other labeling that is directed to or discloses any of the methods or disclosures disclosed herein.

A method of improving the stability of an aqueous pharmaceutical formulation including an anti-human receptor activator of nuclear factor kappa-B ligand (anti-RANKL) human monoclonal antibody or an antigen-binding portion thereof, at a concentration greater than 70 mg/ml, including the step of preparing the aqueous pharmaceutical formulation including the anti-human receptor activator of nuclear factor kappa-B ligand (anti-RANKL) human monoclonal antibody or an antigen-binding portion thereof at a pH in a range of about 5.0 to less than 5.2, wherein the aqueous pharmaceutical formulation demonstrates improved stability at the pH in a range of about 5.0 to less than 5.2 compared to an equivalent aqueous pharmaceutical formulation not at a pH in a range of about 5.0 to less than 5.2.

A method of improving the stability of an aqueous pharmaceutical formulation including an anti-human receptor activator of nuclear factor kappa-B ligand (anti-RANKL) human monoclonal antibody or an antigen-binding portion thereof is disclosed, including the step of preparing the aqueous pharmaceutical formulation comprising the anti-human receptor activator of nuclear factor kappa-B ligand (anti-RANKL) human monoclonal antibody or an antigen-binding portion thereof in admixture with an amino acid aggregation inhibitor, wherein the aqueous pharmaceutical formulation demonstrates improved stability with the amino acid aggregation inhibitor compared to an equivalent aqueous pharmaceutical formulation without the amino acid aggregation inhibitor.

A method of reducing the level of HMWS aggregates in a solution of denosumab or another human anti-RANKL monoclonal antibody is described.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1, 2, and 8 show the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37°C for various high-concentration denosumab formulations. The legend in Figure 1 corresponds to the formulation with the abbreviation shown in Table 1. The legend in Figure 8 corresponds to the letter shown in Table 5.

Figure 3 shows the size exclusion chromatograms for various high-concentration denosumab formulations after storage at 37°C for 1 month. The legend in Figure 3 corresponds to the formulation with the abbreviation shown in Table 2.

Figure 4 is a graph of the % HMWS monitored by SE-UHPLC as a function of time for each formulation having the corresponding F# shown in Table 3A.

Figure 5 is a pair of size exclusion chromatograms for the formulations listed in Table 3A. The legend for Figure 5 corresponds to the Formulation Name listed in Table 3B.

Figure 6 is a graph of % HMWS monitored by SE-UHPLC as a function of storage time at 37 °C for each formulation having the corresponding F# shown in Table 4A.

Figure 7A shows the size exclusion chromatograms for the pH 4.8 formulations having the denosumab concentration listed in Table 4A.

Figure 7B shows the size exclusion chromatograms for the formulations at pH 5.1 having the denosumab concentration listed in Table 4A.

Figure 9 is a graph of % HMWS monitored by SE-UHPLC as a function of storage time at 37 °C for each formulation having the corresponding F# shown in Table 6B.

Figure 10 shows the size exclusion chromatograms as a function of formulation after storage at 37 °C for 1 month for the formulations having the name indicated in Table 6B.

Figure 11A is a graph of the percentage of HMWS monitored by SE-UHPLC as a function of time at 37 °C for the formulation having the letter indicated in Table 7B.

Figure 11B is a graph of the percentage of HMWS monitored by SE-UHPLC as a function of time at 40 °C for the formulation having the letter indicated in Table 7C.

Figure 12A shows the size exclusion chromatograms for the formulations in Table 7B. Figure 12B shows the size exclusion chromatograms for the formulations in Table 7C. Figures 13, 14, and 15 are graphs of the percentage of HMWS monitored by SE-UHPLC as a function of storage time at 37 °C for each formulation having the corresponding Formulation letter shown in Table 8A. Figures 16, 17, and 18 are the chromatographic overlays of the formulations listed in Table 8A after storage at 37 °C for 1 month. Figures 13 and 16 relate to formulations comprising aromatic amino acids, Figures 14 and 17 relate to formulations comprising polar/charged amino acids, and Figures 15 and 18 relate to formulations comprising hydrophobic amino acids.

Figures 19-24 are graphs of % deuterium incorporation at 4 °C as a function of time (log(s)) for light chain amino acids 28-33 (Figure 19), light chain amino acids 108-116 (Figure 20), light chain amino acids 125-132 (Figure 21), heavy chain amino acids 47-59 (Figure 22), heavy chain amino acids 243-253 (Figure 23), and heavy chain amino acids 392-399 (Figure 24) for each of formulations 35-38.

Figures 25-30 are graphs of % deuterium incorporation at 37 °C as a function of time (log (s)) for light chain amino acids 28-33 (Figure 25), light chain amino acids 108-117 (Figure 26), light chain amino acids 124-131 (Figure 27), heavy chain amino acids 47-59 (Figure 28), heavy chain amino acids 242-253 (Figure 29), and heavy chain amino acids 392-399 (Figure 30) for each of formulations 35-38.

Figure 31 is a graph of the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37 °C with the Formulation name indicated in Table 10.

Figure 32 is a graph of the percentage of LMWS as monitored by SE-UHPLC as a function of formulation and time at 37 °C with the Formulation name indicated in Table 11.

Figure 33 is a graph of the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37 °C with the Formulation name indicated in Table 12.

Figure 34 is a graph of the percentage of LMWS as monitored by SE-UHPLC as a function of formulation and time at 37 °C with the Formulation name indicated in Table 13.

Figure 35 is a graph of the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37 °C with the Formulation name indicated in Table 14.

Figure 36 is a graph of the percentage of LMWS as monitored by SE-UHPLC as a function of formulation and time at 37 °C with the Formulation name indicated in Table 15.

Figure 37 are overlays of size exclusion chromatograms for each formulation having the Formulation name indicated in Table 10.

Figure 38 are overlays of size exclusion chromatograms for each formulation having the Formulation name indicated in Table 12.

Figure 39 are overlays of size exclusion chromatograms for each formulation having the Formulation name indicated in Table 14.

Figures 40A and 40B are graphs containing isothermal chemical denaturation curves of denosumab in the absence of arginine, at pH 4.5, 4.8, and 5.0. Figure 40A is a graph of the fraction of denosumab denatured as a function of denaturant concentration. Figure 40B is a graph plotting dF/d[denaturant] as a function of denaturant concentration.

Figures 41A and 41B are graphs containing isothermal chemical denaturation curves of denosumab in the presence of 75 mM arginine HCl at pH 4.5, 4.8, and 5.2. Figure 41A is a graph of the fraction of denosumab denatured as a function of denaturant concentration. Figure 41B is a graph plotting dF/d[denaturant] as a function of denaturant concentration.

Figures 42 and 43 are graphs of the percentage of HMWS monitored by SE-UHPLC as a function of time at 25 °C for 3 months and 37 °C for 2 months, respectively, for the formulation having the formulation name in Table 17.

DETAILED DESCRIPTION

It would be desirable to provide a more concentrated aqueous solution of denosumab, and other human anti-RANKL antibodies, and antigen-binding portions thereof, that are as stable as, or more stable than, diluted solutions. The more concentrated solution could provide patient convenience, for example, by allowing the administration of a smaller volume, such as a 1 ml injection, to deliver 120 mg of active, such as denosumab, rather than a 1.7 ml or 2 ml injection of a more diluted active formulation. Still further, it would allow an even smaller injection volume solution to deliver a lower dose of active, for example, 0.5 ml of 120 mg/ml concentration of denosumab to deliver a 60 mg dose. It would also be desirable to provide an aqueous solution of denosumab, and other human anti-RANKL antibodies, and antigen-binding portions thereof, that are more stable than previously known solutions. The stable, concentrated formulation will also have other benefits, such as allowing for the handling and shipping of smaller volumes of product, and allowing for longer product half-lives.

Aggregates in biologics can vary in origin, size, and type. Of particular concern are aggregates that may affect the efficacy or safety of a biologic, for example, aggregates that may enhance immune responses and cause adverse clinical effects. High molecular weight aggregates, also known as high molecular weight species (HMWS), particularly those that retain most of the native configuration of the monomeric counterpart, may be of particular concern. Aggregation may also potentially affect the subcutaneous bioavailability and pharmacokinetics of a therapeutic protein.

The formation of aggregates can have various causes. In general, protein aggregation results from conformational instability, which is the result of structural changes in proteins, and colloidal instability, which is dominated by intermolecular forces. In the case where a critical nucleation event is required to induce precipitation, protein aggregation kinetics can be characterized by the inclusion of a lag time phase.

Aggregation due to conformational instability involves the steps of unfolding and association. The unfolding of the protein molecule exposes hydrophobic amino acid residues. Hydrophobic residues in unfolded molecules can then undergo association, leading to aggregation (e.g., as dimers, trimers, other multimers, and higher-order aggregates). Such associations are concentration-dependent. An increase in protein concentration in an aqueous solvent generally increases the rate and extent of aggregation, including thermally induced aggregation. Therefore, additives that affect the free energy of protein unfolding in solution can affect conformational stability.

Colloidal instability results in aggregates formed through intramolecular protein-protein association forces. These forces can be affected by one or more factors, including ionic strength, solution pH, and buffer types.

Denosumab is commercially available in solution forms at 60 mg/mL and 70 mg/mL concentrations. Attempts to formulate higher concentrations of denosumab solutions using the same excipients showed that the higher concentration affected product stability, through a concomitant and proportional increase in HMWS. For example, a 120 mg/mL concentration of denosumab is more than 70% higher than 70 mg/mL denosumab and is twice as high as the 60 mg/mL concentration.

Accordingly, a stabilized aqueous formulation according to the present disclosure will resist aggregate formation to a greater degree than previously known formulations. A stabilized aqueous formulation characterized by a pH of 5.0 to less than 5.2 is described. Another non-limiting disclosure is a stabilized aqueous formulation that includes an amino acid aggregation inhibitor. Related dosage forms, for example, as single-use vials, syringes, and glass containers, and related treatment methods are also described. Methods of preparing stable pharmaceutical formulations are further described.

As described below, pH and amino acid aggregation inhibitors (e.g., arginine, arginine-arginine dipeptide, arginine-phenylalanine dipeptide) were shown to reduce the level of HMWS and the rate of HMWS formation of denosumab at 120 mg/mL. HMWS can be described as intermolecular protein interactions that are either irreversible (e.g., covalent) or reversible (e.g., non-covalent self-associated interactions). There are four well-accepted causes of protein self-association reactions that can lead to increases in viscosity and HMWS; hydrophobic, charged, polar, and dipole interactions. Both the pH of the formulation and arginine (a highly charged basic amino acid at neutral to acidic pH values) can interfere with the intermolecular forces of charged proteins. Without wishing to be bound by any particular theory, it is conceivable that the formation of HMWS from denosumab at 120 mg/mL is based on protein loading, and these formulation changes are altering the loading forces involved in the mechanism of HMWS formation. Furthermore, without wishing to be bound by any particular theory, it is conceivable that hydrophobic protein self-association interactions could also be involved in HMWS formation, since arginine contains a short aliphatic hydrocarbon chain in the side chain. This aliphatic chain may alter hydrophobic interactions between proteins. This idea is further supported by the inclusion of phenylalanine in the formulation to further reduce HMWS levels. Without wishing to be bound by any particular theory, arginine stabilizes the anti-RANKL antibody in a different way than phenylalanine, such that if arginine interacts with the antibody through hydrophobic interactions, arginine may interact with the antibody in one or more of the other ways.

Other excipients that may have a potentially positive impact on the level of HMWS reduction and the rate of formation may have a positively charged group similar to neutral to acidic pH values when compared to arginine, and/or may be hydrophobic in nature similar to phenylalanine. Examples of such excipients may include lysine, N-acetylarginine, N-acetyl-lysine, tyrosine, tryptophan, and leucine.

Formulations, dosage forms and methods are contemplated to include any combination of one or more of the additional elements, features and optional steps described below (including those shown in the figures), unless otherwise stated.

In jurisdictions that prohibit patenting methods that are performed on the human body, the meaning of "administering" a composition to a human subject should be restricted to prescribing a controlled substance to be self-administered by a human subject by any technique (e.g., orally, inhalation, topical administration, injection, insertion, etc.). The broadest reasonable interpretation consistent with the laws or regulations defining patentable subject matter is provided. In jurisdictions that do not prohibit patenting methods that are performed on the human body, "administering" compositions includes both methods performed on the human body and the foregoing activities.

As used herein, the term "comprising" indicates the possible inclusion of other agents, elements, steps or features in addition to those specified.

Each maximum numerical limitation given throughout this specification should be understood to include, as alternative aspects, ranges formed with each corresponding lower numerical limitation, as if such ranges were explicitly written. Each minimum numerical limitation given throughout this specification will include, as alternative aspects, ranges formed with each corresponding higher numerical limitation, as if such ranges were explicitly written. Each numerical range given throughout this specification will include each narrower numerical interval that falls within such wider numerical interval, as if such narrower numerical intervals were all explicitly written herein. The dimensions and values disclosed herein should be understood to include disclosure of both the recited value and the corresponding exact numerical value, e.g., a value described as "about 10 mM" should be understood to include, as an alternative disclosure, "10 mM."

The term "therapeutically effective amount," as used herein, refers to an amount of a compound sufficient to treat, ameliorate, or prevent the identified disease or condition, or to have a detectable therapeutic, prophylactic, or inhibitory effect. The effect may be detected, for example, by an improvement in clinical status or a reduction in symptoms. The precise effective amount for a subject will depend on the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. If a drug has been authorized by the U.S. Food and Drug Administration (FDA), a "therapeutically effective amount" refers to the dose authorized by the FDA or its foreign counterpart agency for the treatment of the identified disease or condition.

The present disclosure provides stabilized (or stable) aqueous pharmaceutical formulations as demonstrated by reduced amounts of aggregates and/or reduced rates of aggregate formation upon storage. As described herein, the stability of such formulations is demonstrated by reduced amounts of HMWS and/or reduced rates of HMWS formation upon storage for varying periods of time and at varying temperatures. In general, formulations of greater stability are associated with lower amounts of HMWS, lower rates of HMWS formation, and/or larger major antibody peaks at higher storage temperatures, relative to lower temperatures. As used herein, the term "high molecular weight species" or "HMWS" refers to higher-order aggregates of the antibody in the formulations, as well as lower-order aggregates of the antibody in the formulations. Lower-order aggregates include, for example, dimeric species. The amounts and rates of aggregate formation can be measured or monitored by techniques such as, for example, SE-UHPLC. SE-UHPLC chromatograms of the antibody, in some cases, show a peak around 5.8 minutes representing the amount of HMWS in the aqueous pharmaceutical formulation, a peak around 6.7 minutes representing dimeric species, and a peak around 8.0 minutes reflecting the amount of intact, non-aggregated forms of the antibody. Relative to storage at 4°C, storage at 37°C allows for acceleration of a stability assay so that the stability of a particular formulation can be determined in a shorter period of time, relative to the time period of storage at 4°C. For example, storage at 37°C for 1, 2, or 3 months may be indicative or predictive of storage at 4°C for 36 months.

A stabilized formulation as described herein may exhibit a reduced degree and rate of HMWS formation after 3 months of storage at 37°C, compared to a control formulation of equivalent concentration consisting of 10 mM acetate, 5% (w/v) sorbitol, 0.01% (w/v) polysorbate 20 as excipients and having a solution pH of 5.2.

A stabilized formulation as described herein and including an amino acid aggregation inhibitor can exhibit a reduced degree of HWMS formation after 1 month of storage at 37°C, compared to an equivalent control formulation without the amino acid aggregation inhibitor. For example, the degree of formation can be reduced such that the % amount of HMWS by SE-UPHLC is lower by at least about 0.1%, or about 0.2%, or about 0.3%, or about 0.4%, or about 0.5%, or about 0.6%, or about 0.7%, for example in a range of about 0.1% to about 2%, or about 0.1% to about 1%, compared to the control formulation after 1 month of storage at 37°C.

A stabilized formulation as described herein may have a low amount of HMWS after 1 month of storage at 37°C, by SE-UHPLC. For example, the amount of HMWS may be no more than 2%, or less than 2%, or no more than 1.9%, or less than 1.9%, or no more than 1.8%, or less than 1.8%, or no more than 1.7%, or less than 1.7%, or no more than 1.6%, or less than 1.6%, or no more than 1.5%, or less than 1.5%, or no more than 1.4%, or less than 1.4%, or no more than 1.3%, or less than 1.3%, or no more than 1.2%, or less than 1.2%, for example, in a range of about 0.01% to about 2%, or about 0.01% to about 1.9%, or about 0.01% to about 1.8%, or about 0.01% to about 1.7%, or about 0.01% to about 1.6%, or about 0.01% to about 1.5%, or about 0.01% to about 1.4%, or about 0.01% to about 1.3%, or about 0.01% to about 1.2%. The amount of HMWS after 1 month storage at 37°C, by SE-UHPLC, may be greater than 2%, for example, greater than 2% and up to 3%, while the reduced rate of aggregation provided by the amino acid aggregation inhibitor may allow adequate product shelf stability, for example up to three years, or up to two years.

A stabilized formulation as described herein may have a low amount of HMWS after 3 months of storage at 37°C, by SE-UHPLC. For example, the amount of HMWS may be no more than 2%, or less than 2%, or no more than 1.9%, or less than 1.9%, or no more than 1.8%, or less than 1.8%, or no more than 1.7%, or less than 1.7%, or no more than 1.6%, or less than 1.6%, or no more than 1.5%, or less than 1.5%, or no more than 1.4%, or less than 1.4%, or no more than 1.3%, or less than 1.3%, or no more than 1.2%, or less than 1.2%, for example, in a range of about 0.01% to about 2%, or about 0.01% to about 1.9%, or about 0.01% to about 1.8%, or about 0.01% to about 1.7%, or about 0.01% to about 1.6%, or about 0.01% to about 1.5%, or about 0.01% to about 1.4%, or about 0.01% to about 1.3%, or about 0.01% to about 1.2%.

A stabilized formulation as described herein may have a low amount of HMWS after 36 months of storage at 4°C, by SE-UHPLC. For example, the amount of HMWS may be no more than 2%, or less than 2%, or no more than 1.9%, or less than 1.9%, or no more than 1.8%, or less than 1.8%, or no more than 1.7%, or less than 1.7%, or no more than 1.6%, or less than 1.6%, or no more than 1.5%, or less than 1.5%, or no more than 1.4%, or less than 1.4%, or no more than 1.3%, or less than 1.3%, or no more than 1.2%, or less than 1.2%, for example, in a range of about 0.01% to about 2%, or about 0.01% to about 1.9%, or about 0.01% to about 1.8%, or about 0.01% to about 1.7%, or about 0.01% to about 1.6%, or about 0.01% to about 1.5%, or about 0.01% to about 1.4%, or about 0.01% to about 1.3%, or about 0.01% to about 1.2%.

A stabilized formulation as described herein may have a high amount of the major peak of denosumab or other antibody (or antigen-binding portion thereof) after 1 month of storage at 37°C, by SE-UHPLC. For example, the amount of the major peak may be at least 95%, or greater than 95%, or at least 96%, or greater than 96%, or at least 97%, or greater than 97%, or at least 97.5%, or greater than 97.5%, or at least 98%, or greater than 98%, or at least 98.1%, or greater than 98.1%, or at least 98.2%, or greater than 98.2%, or at least 98.3%, or greater than 98.3%, or at least 98.4%, or greater than 98.4%, or at least 98.5%, or greater than 98.5%, or at least 98.6%, or greater than 98.6%, for example in a range of about 95% to about 99.9%, or about 96% to about 99.9%, or about 97% to about or about 98.6% to about 99.9%.

A stabilized formulation as described herein may have a high amount of the major peak of denosumab or other antibody (or antigen-binding portion thereof) after 3 months of storage at 37°C, by SE-UHPLC. For example, the amount of the major peak may be at least 95%, or greater than 95%, or at least 96%, or greater than 96%, or at least 97%, or greater than 97%, or at least 97.5%, or greater than 97.5%, or at least 98%, or greater than 98%, or at least 98.1%, or greater than 98.1%, or at least 98.2%, or greater than 98.2%, or at least 98.3%, or greater than 98.3%, or at least 98.4%, or greater than 98.4%, or at least 98.5%, or greater than 98.5%, or at least 98.6%, or greater than 98.6%, for example, in a range of about 95% to about 99.9%, or about 96% to about 99.9%, or about 97% to about 99.9%, or about 97.5% to about 99.9%, or about 98% to about 99.9%, or about 98.1% to about 99.9%, or about 98.2% to about 99.9%, or about 98.3% to about 99.9%, or about 98.4% to about 99.9%, or about 98.5% to about 99.9%, or about 98.6% to about 99.9%.

A stabilized formulation as described herein may have a high amount of the major peak of denosumab or other antibody (or antigen-binding portion thereof) after 36 months of storage at 4°C, by SE-UHPLC. For example, the amount of the major peak may be at least 95%, or greater than 95%, or at least 96%, or greater than 96%, or at least 97%, or greater than 97%, or at least 97.5%, or greater than 97.5%, or at least 98%, or greater than 98%, or at least 98.1%, or greater than 98.1%, or at least 98.2%, or greater than 98.2%, or at least 98.3%, or greater than 98.3%, or at least 98.4%, or greater than 98.4%, or at least 98.5%, or greater than 98.5%, or at least 98.6%, or greater than 98.6%, for example, in a range of about 95% to about 99.9%, or about 96% to about 99.9%, or about 97% to about 99.9%, or about 97.5% to about 99.9%, or about 98% to about 99.9%, or about 98.1% to about 99.9%, or about 98.2% to about 99.9%, or about 98.3% to about 99.9%, or about 98.4% to about 99.9%, or about 98.5% to about 99.9%, or about 98.6% to about 99.9%.

It is contemplated that the stabilized formulation may have either a low amount of HMWS or a high amount of main peak, according to a specification described above, upon storage.

The aqueous pharmaceutical formulations may comprise no more than about 4% high molecular weight species (HMWS) and/or may comprise greater than about 96% of the major antibody peak, as measured by SE-UHPLC, upon storage. The aqueous pharmaceutical formulations may comprise no more than about 3% high molecular weight species (HMWS) and/or may comprise greater than about 97% of the major antibody peak, as measured by SE-UHPLC, upon storage. The aqueous pharmaceutical formulations may comprise less than about 2% HMWS and/or greater than about 98% of the major antibody peak, as measured by SE-UHPLC, upon storage. Storage may be at a temperature of about 2°C to about 8°C (e.g., about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C) for at least 12 months, 24 months, or 36 months (e.g., at least or about 12 months, at least or about 16 months, at least or about 20 months, at least or about 24 months, at least or about 28 months, at least or about 32 months, at least or about 36 months, optionally, longer). Storage may be at about 20°C to about 30°C (e.g., about 21°C to about 30°C, about 22°C to about 30°C, about 23°C to about 30°C, about 24°C to about 30°C, about 25°C to about 30°C, about 26°C to about 30°C, about 27°C to about 30°C, about 28°C to about 30°C, about 28°C to about 30°C, about 20°C to about 29°C, about 20°C to about 28°C, about 20°C to about 27°C, about 20°C to about 26°C, about 20°C to about 25°C, about 20°C to about 24°C, about 20°C to about 23°C). °C, about 20 °C to about 22 °C) for about 1 month (e.g., about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 36 days). The storage may comprise a first storage followed by a second storage, wherein the first storage is from about 2 °C to about 8 °C for at least 12 months, 24 months, or 36 months and the second storage is from about 20 °C to about 30 °C for about 1 month. The aqueous pharmaceutical formulations may comprise no more than 2% HMWS, or less than 2% HMWS, or no more than 1.9% HMWS, or less than 1.9% HMWS, or no more than 1.8% HMWS, or less than 1.8% HMWS, or no more than 1.7% HMWS, or less than 1.7% HMWS, or no more than 1.6% HMWS, or less than 1.6% HMWS, or no more than 1.5% HMWS, or less than 1.5% HMWS, or no more than 1.4% HMWS, or less than 1.4% HMWS, or no more than 1.3% HMWS, or less than 1.3% HMWS, or no more than 1.2% HMWS, or less than 1.2% HMWS, for example in a range of about 0.01% to about 2% HMWS, or about 0.01% to about 1.9% HMWS, or about 0.01% to about 1.8% HMWS, or about 0.01% to about 1.7% HMWS, or about 0.01% to about 1.6% HMWS, or about 0.01% to about 1.5% HMWS, or about 0.01% to about 1.4% HMWS, or about 0.01% to about 1.3% HMWS, or about 0.01% to about 1.2% HMWS, optionally, as measured by SE-UHPLC. The aqueous pharmaceutical formulations may comprise more than 98% of the major antibody peak, or at least 95% of the major antibody peak, or more than 95% of the major antibody peak, or at least 96% of the major antibody peak, or more than 96% of the major antibody peak, or at least 97% of the major antibody peak, or more than 97% of the major antibody peak, or at least 97.5% of the major antibody peak, or more than 97.5% of the major antibody peak, or at least 98% of the major antibody peak, or more than 98% of the major antibody peak, or at least 98.1% of the major antibody peak, or more than 98.1% of the major antibody peak, or at least 98.2% of the major antibody peak, or more than 98.2% of the major antibody peak, or at least 98.3% of the major antibody peak, or more than 98.3% of the major antibody peak. antibody, or at least 98.4% of the major antibody peak, or more than 98.4% of the major antibody peak, or at least 98.5% of the major antibody peak, or more than 98.5% of the major antibody peak, or at least 98.6% of the major antibody peak, or more than 98.6% of the major antibody peak, for example in a range of about 95% to about 99.9% of the major antibody peak, or about 96% to about 99.9% of the major antibody peak, or about 97% to about 99.9% of the major antibody peak, or about 97.5% to about 99.9% of the major antibody peak, or about 98% to about 99.9% of the major antibody peak, or about 98.1% to about 99.9% of the major antibody peak, or about 98.2% to about 99.9% of the major antibody peak, or about 98.3% to about 99.9% of the major antibody peak, or about 98.4% to about 99.9% of the major antibody peak, or about 98.5% to about 99.9% of the major antibody peak, or about 98.6% to about 99.9% of the major antibody peak, optionally, as measured by SE-UHPLC.

As used herein, the term "antibody" refers to a protein having a conventional immunoglobulin format, comprising heavy and light chains, and comprising variable and constant regions. For example, an antibody may be an IgG which is a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair having a "light" chain (typically having a molecular weight of about 25 kDa) and a "heavy" chain (typically having a molecular weight of about 50-70 kDa). An antibody has both a variable region and a constant region. In IgG formats, the variable region is generally about 100-110 or more amino acids, comprises three complementarity-determining regions (CDRs), is primarily responsible for antigen recognition, and varies substantially among other antibodies that bind to different antigens. See, for example, Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes," Immunobiology: The Immune System in Health and Disease, 4th ed. Elsevier Science Ltd./Garland Publishing, (1999).

Briefly, in an antibody scaffold, the CDRs are embedded within a framework in the heavy and light chain variable regions where they constitute the regions largely responsible for antigen binding and recognition. A variable region comprises at least three heavy chain CDRs or three light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883), within a framework region (framework regions designated 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra).

Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. The disclosures described herein include all such antibody classes or isotypes. The light chain constant region may be, for example, a kappa or lambda light chain constant region, e.g., a human kappa or lambda light chain constant region. The heavy chain constant region may be, for example, an alpha, delta, epsilon, gamma, or mu type heavy chain constant region, for example a human alpha, delta, epsilon, gamma, or mu type heavy chain constant region. Accordingly, the antibody may be an antibody of IgA, IgD, IgE, IgG, or IgM isotype, including any one of IgG1, IgG2, IgG3, or IgG4. The anti-RANKL antibody may be an IgG1, IgG2, or IgG4 antibody.

The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody may comprise a sequence that is substantially similar to a naturally occurring antibody produced by a mammal, e.g., mouse, rat, rabbit, goat, horse, chicken, hamster, pig, or human. In this regard, the antibody may be considered a mammalian antibody, e.g., a mouse antibody, rat antibody, rabbit antibody, goat antibody, horse antibody, chicken antibody, hamster antibody, pig antibody, or human antibody. The anti-RANKL antibody may be a human monoclonal antibody. The recombinant protein may be a chimeric antibody or a humanized antibody. The term "chimeric antibody" is used herein to refer to an antibody that contains constant domains from one species and the variable domains of a second, or more, generally containing stretches of amino acid sequence from at least two species. The term "humanized," when used with respect to antibodies, refers to antibodies that have at least CDR regions from a non-human source that are engineered to have a structure and immunological function more similar to true human antibodies than antibodies from the original source. For example, humanizing may involve grafting CDRs from a non-human antibody, such as a mouse antibody, onto a human antibody. Humanizing may also involve selecting amino acid substitutions to make a non-human sequence more human-like.

An antibody can be cleaved into fragments by enzymes, such as, for example, papain and pepsin. Papain cleaves an antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves an antibody to produce an F(ab<')2> fragment and a pFc' fragment. The aqueous pharmaceutical formulation can comprise an antibody fragment, for example, a Fab, Fc, F(ab<')2>, or pFc', that retains at least one antigen-binding site (RANKL). With respect to the aqueous pharmaceutical formulations and methods of the present disclosure, the antibody can lack certain portions of an antibody, and can be an antibody fragment that binds RANKL. The antibody fragment can be an antigen-binding portion of an anti-RANKL antibody.

Antibody protein products can be an antigen-binding format based on antibody fragments, for example, scFv, Fab, and VHH/VH, which retain full antigen-binding capacity. The smallest antigen-binding fragment that retains its complete antigen-binding site is the Fv fragment, which consists entirely of variable (V) regions. A soluble and flexible amino acid peptide linker is used to connect the V regions with a scFv (single-chain variable fragment) fragment for stabilization of the molecule, or constant (C) domains are added to the V regions to generate a Fab fragment (fragment, antigen binding). Both scFv and Fab are widely used fragments that can be easily produced in hosts, for example, prokaryotic hosts. Other antibody protein products include disulfide-bond-stabilized scFv (ds-scFv), single-chain Fab (scFab), as well as di- and multimeric antibody formats such as dia-, tria- and tetra-bodies, or minibodies (miniAbs) comprising different formats consisting of the scFvs linked to oligomerization domains. The smallest fragments are V/V fragments of camelid heavy chain Abs as well as single-domain Abs (sdAbs). The most frequently used structural element to create novel antibody formats is the single-chain variable domain (V) antibody fragment (scFv), which comprises the heavy and light chain V domains (V and V domains) joined by a peptide linker of ~15 amino acid residues. A peptibody or peptide-Fc fusion is yet another antibody protein product. The structure of a peptibody consists of a biologically active peptide grafted onto an Fc domain. Peptibodies are well described in the art. See, for example, Shimamoto et al., mAbs 4(5): 586–591 (2012).

Other antibody protein products include, for example, a single-chain antibody (SCA); a diabody; a triabody; a tetrabody; and bispecific or trispecific antibodies. Bispecific antibodies can be divided into five major classes: BsIgG, appended IgG, BsAb fragments, bispecific fusion proteins, and BsAb conjugates. See, for example, Spiess et al., Molecular Immunology 67(2) Part A: 97–106 (2015).

The anti-RANKL antibody, or antigen-binding portion thereof, may comprise, consist essentially of, or consist of any one of these antibody protein products (e.g., scFv, VHH/VH Fab, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., a diabody, triabody, tetrabody), miniAb, camelid heavy chain antibody VHH/VH peptibody, sdAb, diabody; a triabody; a tetrabody; a bispecific or trispecific antibody, BsIgG, linked IgG, BsAb fragment, bispecific fusion protein, and BsAb conjugate).

The anti-RANKL antibody, or antigen-binding portion thereof, may comprise, consist essentially of, or consist of an antibody protein product in monomeric form, or polymeric, oligomeric, or multimeric form. In certain embodiments, where the antibody comprises two or more distinct antigen-binding region fragments, the antibody is considered bispecific, trispecific, or multispecific, or bivalent, trivalent, or multivalent, depending on the number of distinct epitopes that are recognized and bound by the antibody.

A human anti-receptor activator of nuclear factor kappa-B ligand (anti-RANKL) antibody or antigen-binding portion thereof for use in the formulation is an antibody or antigen-binding portion thereof that specifically binds to human RANKL protein or human osteoprotegrin (OPGL) protein or a fragment thereof and inhibits or neutralizes the activity of RANKL or OPGL protein and/or inhibits the RANK/RANKL signaling pathway, and is referred to herein as a human anti-RANKL monoclonal antibody or antigen-binding portion thereof. For example, the formulations described herein may comprise a human anti-RANKL monoclonal antibody that specifically binds to the amino acid sequence of human RANKL (SEQ ID NO: 12) or a portion thereof. The human RANKL protein is a transmembrane or soluble protein encoded by the polynucleotide sequence SEQ ID NO: 11, which is known to be essential for osteoclast formation, function, and survival. For example, human anti-RANKL antibodies inhibit the interaction of RANKL with its receptor, RANK.

An example of a human anti-RANKL monoclonal antibody is denosumab, which is marketed commercially as Xgeva® and Prolia®. Xgeva® is a 120 mg dose in 1.7 mL solution (70 mg/mL) denosumab formulation in a single-use vial, containing 120 mg of denosumab, acetate (18 mM), sorbitol (4.6%), water for injection (USP), and sodium hydroxide at a pH of 5.2. Prolia® is available as 60 mg dose in 1 mL solution (60 mg/mL) denosumab formulations. Each 1 mL single-use prefilled syringe of Prolia® contains 60 mg of denosumab (60 mg/mL solution), 4.7% sorbitol, 17 mM acetate, 0.01% polysorbate 20, Water for Injection (USP), and sodium hydroxide at a pH of 5.2. Specifically contemplated are formulations described herein that include denosumab or a portion thereof. Denosumab is a fully human IgG2 monoclonal antibody that binds to human RANKL. Denosumab has an approximate molecular weight of 147 kDa and is expressed in the Chinese hamster ovary (CHO) cell line. The amino acid sequences of the variable light chain (LC) and variable heavy chain (HC) of denosumab are set forth in SEQ ID NOs: 1 and 2, respectively, and the full-length LC and HC are set forth as SEQ ID NOs: 3 and 4, respectively. A nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 (denosumab variable LC) is, optionally, a nucleic acid of SEQ ID NO: 19. A nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 (denosumab variable HC) is, optionally, a nucleic acid of SEQ ID NO: 20. A nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 (denosumab full-length LC) is, optionally, a nucleic acid of SEQ ID NO: 21. A nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4 (denosumab full-length HC) is, optionally, a nucleic acid of SEQ ID NO: 23. The mature form of LC, which is represented as amino acids 21-235 of full-length LC, is set forth as SEQ ID NO: 13, while the mature form of HC, which is represented as amino acids 20-467 of full-length HC, is set forth as SEQ ID NO: 14. A nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 13 (the mature form of LC) is, optionally, a nucleic acid of SEQ ID NO: 22. A nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14 (the mature form of HC) is, optionally, a nucleic acid of SEQ ID NO: 24. Additionally, CDRs of denosumab LC are set forth as SEQ ID NO: 5 (LC<c>D<r>1), SEQ ID NO: 6 (LC CDR2), and SEQ ID NO: 7 (LC CDR3). The CDRs of denosumab HC are set forth as SEQ ID NO: 8 (HC CDR1), SEQ ID NO: 9 (HC CDR2), and SEQ ID NO: 10 (HC CDR3). Denosumab has been described and claimed in International Patent Application No. WO 03/002713 and U.S. Patent No. 27,364,736.

As used herein, the term "denosumab" includes biosimilars of denosumab. As used herein, "biosimilar" (of an authorized reference product/biological drug, such as a therapeutic protein, antibody, etc.) refers to a biological product that is similar to the reference product based on data derived from (a) analytical studies demonstrating that the biological product is highly similar to the reference product despite minor differences in clinically inactive components; (b) animal studies (including toxicity assessment); and/or (c) a clinical study or studies (including immunogenicity and pharmacokinetic or pharmacodynamic assessment) that are sufficient to demonstrate safety, purity, and potency under one or more appropriate conditions of use for which the reference product is licensed and intended to be used and for which a license is sought for the biological product. The biosimilar biological product and the reference product may use the same mechanism(s) of action for the condition(s) of use prescribed, recommended, or suggested in the proposed labeling, but only to the extent that the mechanism(s) of action are known for the reference product. The condition(s) of use prescribed, recommended, or suggested in the proposed labeling for the biological product may have been previously authorized for the reference product. The route of administration, dosage form, and/or strength of the biological product may be the same as those of the reference product. The ease with which the biological product is manufactured, processed, packaged, or maintained may meet standards designed to ensure that the biological product remains safe, pure, and potent. The reference product may be authorized in at least one of the U.S., Europe, or Japan. A biosimilar may be, for example, an antibody that has the same primary amino acid sequence as a marketed antibody, but may be prepared in different cell types or by different production, purification, or formulation methods.

The formulations may comprise a human anti-RANKL antibody comprising at least one of the amino acid sequences of SEQ ID NO: 1-4, 13, 14, or a portion thereof. The formulations may comprise a human anti-RANKL antibody comprising at least one of the CDR amino acid sequences set forth as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or at least two of the CDR amino acid sequences set forth as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or at least three of the CDR amino acid sequences set forth as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or at least four of the CDR amino acid sequences set forth as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, or at least five of the CDR amino acid sequences set forth as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, or at least six of the CDR amino acid sequences set forth as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

The formulations may comprise an anti-human RANKL antibody comprising at least one amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 1-4, 13, and 14 and inhibits the interaction between RANKL and its receptor, RANK, or an anti-human RANKL antibody comprising at least one amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 1-4, 13, and 14 and inhibits the interaction between RANKL and its receptor, RANK, or an anti-human RANKL antibody comprising at least one amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 1-4, 13, and 14 and inhibits the interaction between RANKL and its receptor, RANK, or an anti-human RANKL antibody comprising at least one amino acid sequence that is at least 91% identical to any one of SEQ ID NOs: 1-4, 13, and 14 and inhibits the interaction between RANKL and its receptor, RANK, or a human anti-RANKL antibody comprising at least one amino acid sequence that is at least 92% identical to any one of SEQ ID NOs: 1-4, 13, and 14 and inhibits the interaction between RANKL and its receptor, RANK, or a human anti-RANKL antibody comprising at least one amino acid sequence that is at least 93% identical to any one of SEQ ID NOs: 1-4, 13, and 14 and inhibits the interaction between RANKL and its receptor, RANK, or an human anti-RANKL antibody comprising at least one amino acid sequence that is at least 94% identical to any one of SEQ ID NOs: 1-4, 13, and 14 and inhibits the interaction between RANKL and its receptor, RANK, or an human anti-RANKL antibody comprising at least one amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 1-4, 13, and 14 and inhibits the interaction between RANKL and its receptor, RANK, or an anti-human RANKL antibody comprising at least one amino acid sequence that is at least 96% identical to any one of SEQ ID NOs: 1-4, 13, and 14 and inhibits the interaction between RANKL and its receptor, RANK, or an anti-human RANKL antibody comprising at least one amino acid sequence that is at least 97% identical to any one of SEQ ID NOs: 1-4, 13, and 14 and inhibits the interaction between RANKL and its receptor, RANK, or an anti-human RANKL antibody comprising at least one amino acid sequence that is at least 98% identical to any one of SEQ ID NOs: 1-4, 13, and 14 and inhibits the interaction between RANKL and its receptor, RANK, or an anti-human RANKL antibody comprising at least one amino acid sequence that is at least 99% identical to any one of SEQ ID NOs: 1-4, 13, and 14 and inhibits the interaction between RANKL and its receptor, RANK.

The aqueous pharmaceutical formulation may comprise an anti-RANKL antibody, or an antigen-binding portion thereof, including an antibody protein product, as described herein. The anti-RANKL antibody, or antigen-binding portion thereof, comprises a light chain variable domain comprising a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO: 5. The anti-RANKL antibody, or antigen-binding portion thereof, may comprise a light chain variable domain comprising a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO: 6. The anti-RANKL antibody, or antigen-binding portion thereof, may comprise a heavy chain variable domain comprising a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO: 10. The anti-RANKL antibody, or antigen-binding portion thereof, may comprise SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 10. The anti-RANKL antibody, or antigen-binding portion thereof, may comprise (i) a light chain variable domain comprising a light chain CDR3 sequence comprising the sequence of amino acids set forth in SEQ ID NO:7; (ii) a heavy chain variable domain comprising a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO: 8, optionally, SEQ ID NO: 27; (iii) a heavy chain variable domain comprising a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO: 9, or (iv) any combination thereof. The anti-RANKL antibody, or antigen-binding portion thereof, may comprise (A) a light chain variable domain comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain variable domain comprising a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable domain comprising a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:7; and (B) a heavy chain variable domain comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8 (optionally, SEQ ID NO: 27), a heavy chain variable domain comprising a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a heavy chain variable domain comprising a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10. The anti-RANKL antibody, or antigen-binding portion thereof, may comprise: (A) a light chain variable domain selected from the group consisting of: (i) a light chain variable domain comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to the anti-RANKL antibody. (ii) a light chain variable domain comprising an amino acid sequence encoded by a polynucleotide sequence comprising SEQ ID NO: 19; and (iii) a light chain variable domain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO: 19; or (B) the heavy chain variable domain selected from the group consisting of: (i) a heavy chain variable domain comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 2; (ii) a heavy chain variable domain comprising an amino acid sequence encoded by a polynucleotide sequence comprising SEQ ID NO: 20, and (iii) a heavy chain variable domain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO: 20; or (C) a light chain variable domain of (A) and a heavy chain variable domain of (B). The anti-RANKL antibody may be a fully human antibody, a humanized antibody, or a chimeric antibody. The antigen-binding portion may be a Fab, Fab', F(ab')2, or a single-chain Fv. The anti-RANKL antibody may be an IgG<1>, IgG<2>, or IgG4 antibody, optionally, wherein the anti-RANKL antibody comprises a sequence of SEQ ID NO: 15. The anti-RANKL antibody may comprise a sequence of SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. The anti-RANKL antibody, or antigen-binding portion thereof, may comprise: (A) a light chain selected from the group consisting of: (i) a light chain comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 3 or SEQ ID NO: 13; (ii) a light chain comprising an amino acid sequence encoded by a polynucleotide sequence of SEQ ID NO: 21 or 23; and (iii) a light chain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO: 21 or 23; or (B) a heavy chain selected from the group consisting of: (i) a heavy chain comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 4 or SEQ ID NO: 14; (ii) a heavy chain comprising an amino acid sequence encoded by a polynucleotide sequence of SEQ ID NO: 22 or 24, and (iii) a heavy chain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO: 22 or 24; or (C) a light chain variable domain of (A) and a heavy chain variable domain of (B).

The concentration of denosumab or other human anti-RANKL antibody, or antigen-binding portion thereof, in the aqueous formulation can generally be in any useful range, for example, about 0.1 to about 200 mg/mL. As the concentration increases, there is an increase in viscosity, which may prevent processing of the formulation into a sterile dosage form for pharmaceutical use.

The enhanced stability of the formulation by an amino acid aggregation inhibitor can exist at any concentration of denosumab or other human anti-RANKL antibody, or antigen-binding portion thereof, including about 10 mg/ml to about 200 mg/ml, or about 15 mg/ml to about 150 mg/ml, or about 30 mg/ml to about 200 mg/ml, or about 60 mg/ml to about 200 mg/ml, or about 60 mg/ml to about 180 mg/ml, or about 60 mg/ml to about 160 mg/ml, or about 60 mg/ml to about 150 mg/ml, or about 60 mg/ml to about 140 mg/ml, or about 60 mg/ml to about 130 mg/ml, or about 60 mg/ml to about 120 mg/ml, or about 60 mg/ml to about 110 mg/ml, or about 60 mg/ml to about 100 mg/ml, or about 60 mg/ml to about 90 mg/ml, or about 60 mg/ml to about 80 mg/ml, or about 60 mg/ml to about 70 mg/ml, or about 70 mg/ml to about 200 mg/ml, or about 70 mg/ml to about 180 mg/ml, or about 70 mg/ml to about 160 mg/ml, or about 70 mg/ml to about 150 mg/ml, or about 70 mg/ml to about 140 mg/ml, or about 70 mg/ml to about 130 mg/ml, or about 70 mg/ml to about 120 mg/ml, or about 70 mg/ml to about 110 mg/ml, or about 70 mg/ml to about 100 mg/ml, or about 70 mg/ml to approximately 90 mg/ml, or about 70 mg/ml to about 80 mg/ml, for example 120 mg/ml.

It may be contemplated that the concentration of denosumab or other human anti-RANKL antibody, or antigen-binding portion thereof, for formulations having a pH of about 5.0 to less than 5.2 include ranges greater than 70 mg/ml, or at least about 71 mg/ml, or at least about 75 mg/ml, or at least about 80 mg/ml, or at least about 85 mg/ml, or at least about 90 mg/ml, or at least about 95 mg/ml, or at least about 100 mg/ml, or at least about 105 mg/ml, or at least about 110 mg/ml, or at least about 115 mg/ml, or at least about 120 mg/ml, and up to about 200 mg/ml. For example, contemplated ranges include 71 mg/ml to about 200 mg/ml, or about 75 mg/ml to about 200 mg/ml, or about 75 mg/ml to about 180 mg/ml, or about 75 mg/ml to about 160 mg/ml, or about 75 mg/ml to about 150 mg/ml, or about 75 mg/ml to about 140 mg/ml, or about 75 mg/ml to about 130 mg/ml, or about 75 mg/ml to about 120 mg/ml, or about 75 mg/ml to about 110 mg/ml, or about 75 mg/ml to about 100 mg/ml, or about 75 mg/ml to about 90 mg/ml, or about 120 mg/ml to about 200 mg/ml, or about 120 mg/ml to about 180 mg/ml, or about 120 mg/ml to about 160 mg/ml, or about 120 mg/ml to about 140 mg/ml, for example 120 mg/ml.

The aqueous pharmaceutical formulation may comprise the antibody, or antigen-binding portion thereof, at a concentration greater than 70 mg/ml, for example, greater than 80 mg/ml, greater than 90 mg/ml, greater than 100 mg/ml, greater than 125 mg/ml, greater than 150 mg/ml, greater than 175 mg/ml, greater than 200 mg/ml, greater than 225 mg/ml, greater than 250 mg/ml, greater than 275 mg/ml. The aqueous pharmaceutical formulation may comprise the antibody, or antigen-binding portion thereof, at a concentration of less than about 300 mg/ml, for example, less than about 275 mg/ml, less than about 250 mg/ml, less than about 225 mg/ml, less than about 200 mg/ml, less than about 175 mg/ml, or less than about 150 mg/ml. The concentration of the antibody, or antigen-binding portion thereof, in the formulation is in a range of about 10 mg/ml to about 300 mg/ml, for example, about 25 mg/ml to about 300 mg/ml, about 50 mg/ml to about 300 mg/ml, about 75 mg/ml to about 300 mg/ml, about 125 mg/ml to about 300 mg/ml, about 150 mg/ml to about 300 mg/ml, about 175 mg/ml to about 300 mg/ml, about 200 mg/ml to about 300 mg/ml, about 225 mg/ml to about 300 mg/ml, about 250 mg/ml to about 300 mg/ml, about 275 mg/ml to about 300 mg/ml, about 10 mg/ml to about 275 mg/ml, about 10 mg/ml to approximately 250 mg/ml, approximately 10 mg/ml to approximately 225 mg/ml, approximately 10 mg/ml to approximately 200 mg/ml, approximately 10 mg/ml to approximately 175 mg/ml, approximately 10 mg/ml to approximately 150 mg/ml, approximately 10 mg/ml to approximately 125 mg/ml, approximately 10 mg/ml to approximately 100 mg/ml, approximately 10 mg/ml to about 75 mg/ml, about 10 mg/ml to about 50 mg/ml, or about 10 mg/ml to about 25 mg/ml. The aqueous pharmaceutical formulation may comprise a concentration of the antibody or antigen-binding portion thereof, in a range of greater than 70 mg/ml to about 300 mg/ml, for example, greater than 80 mg/ml to about 300 mg/ml, greater than 90 mg/ml to about 300 mg/ml, greater than 100 mg/ml to about 300 mg/ml, greater than 125 mg/ml to about 300 mg/ml, greater than 150 mg/ml to about 300 mg/ml, greater than 175 mg/ml to about 300 mg/ml, greater than 200 mg/ml to about 300 mg/ml, greater than 70 mg/ml to about 275 mg/ml, greater than about 70 mg/ml to about 250 mg/ml, greater than about 70 mg/ml to about 225 mg/ml, greater than about 70 mg/ml to about 200 mg/ml, greater than about 70 mg/ml to about 175 mg/ml, greater than about 70 mg/ml to about 150 mg/ml, greater than about 70 mg/ml to about 125 mg/ml, greater than about 70 mg/ml to about 100 mg/ml. The aqueous pharmaceutical formulation may comprise a concentration of the antibody or antigen-binding portion thereof in a range of about 100 to about 140 mg/ml, for example, about 110 mg/ml, about 120 mg/ml, about 130 mg/ml. The aqueous pharmaceutical formulation may comprise a concentration of the antibody or antigen-binding portion thereof that is about 120 mg/ml ± 12 mg/ml, for example, about 108 mg/ml to about 132 mg/ml, about 115 mg/ml to about 125 mg/ml, about 116 mg/ml, about 117 mg/ml, about 118 mg/ml, about 119 mg/ml, about 120 mg/ml, about 121 mg/ml, about 122 mg/ml, about 123 mg/ml, about 124 mg/ml.

Denosumab and other human anti-RANKL monoclonal antibodies, and antigen-binding portions thereof, can be prepared according to the description provided in International Patent Publication WO 2003002713 A2.

Formulation studies in high concentration denosumab solutions (e.g., 120 mg/mL), described below, showed a large increase in HMWS formation (rate and extent) below pH 5 and especially at lower pH (e.g., pH 4.5). As the pH increased, there was shown to be an increase in the formation of the dimer species. Balancing the two effects, it is contemplated that a formulation described herein will have a pH in a range of about 5.0 to less than 5.2, or about 5.0 to about 5.19, or about 5.0 to about 5.15, or about 5.0 to about 5.10, e.g., about 5.0, about 5.05, about 5.1, or about 5.15.

The studies described herein also showed an independent stabilizing and aggregation-reducing effect made possible by the inclusion of an amino acid aggregation inhibitor. Accordingly, it is contemplated that when an amino acid aggregation inhibitor is included, the pH of the formulation may be in a range of about 4.9 to about 5.4, or about 5.0 to about 5.4, or about 5.0 to about 5.2, or about 5.0 to less than 5.2, or about 5.0 to 5.19, or about 5.0 to about 5.15, or about 5.0 to about 5.10, e.g., about 5.0, about 5.05, about 5.1, or about 5.15, or about 5.2.

The aqueous formulation may be buffered. When used, the buffer may be an organic buffer. The buffer system may be centered at 25°C around pH 4 to 5.5, or 4.5 to 5.5, or 4.5 to 5, for example. For example, the buffer system may have a pKa within one pH unit of pH 5.0-5.2 at 25°C. Such a buffer system is acetic acid/acetate, which has a pKa of about 4.75 at 25°C. Another such buffer system is glutamic acid/glutamate, which has a pKa of about 4.27 at 25°C. Other contemplated alternative buffer systems include ion-based systems including succinate (pKa of 4.21 at 25°C), propionate (pKa of 4.87 at 25°C), malate (pKa of 5.13 at 25°C), pyridine (pKa of 5.23 at 25°C), and piperazine (pKa of 5.33 at 25°C). It is contemplated that the buffer may be provided as the sodium salt (or disodium salt, as appropriate), or alternatively as a potassium, magnesium, or ammonium salt. The buffers may be based on acetate, citrate, succinate, phosphate, and hydroxymethylaminomethane (Tris), for example. Particularly contemplated are buffers based on acetate, glutamate, and succinate, for example, acetate or glutamate.

A comparison of HMWS formation by size exclusion ultra-high performance liquid chromatography (SE-UHPLC) in 120 mg/mL denosumab formulations having acetate or glutamate buffers, but otherwise equivalent, showed no difference between buffer types when evaluated over four weeks of storage at 37°C.

When used, the buffer will be included in an amount sufficient to maintain the selected pH of the formulation under storage conditions for shelf stability of the product, for example, 3 years at 4°C, or 1 month at 25°C, or 2 weeks at 25°C, or 7 days at 25°C. The buffer concentration may be in a range of about 2 mM to about 40 mM, or about 5 mM to about 20 mM, or about 10 mM to about 25 mM, or about 15 mM to about 25 mM, for example 10 mM, or 15 mM, or 18 mM, or 25 mM. For example, an acetate buffer used with the anti-RANKL monoclonal antibody (e.g., denosumab) and phenylalanine may be in a range of about 2 mM to about 30 mM, or about 16 mM to about 41 mM, or about 25 mM to about 39 mM, or about 30 mM to about 34 mM. In other words, a diafiltration buffer used to concentrate the antibody to a concentration greater than 70 mg/ml (e.g., 120 mg/ml) may be in a range of 5 mM to about 30 mM, or about 15 mM to about 25 mM, or about 20 mM. It is also contemplated to provide an amino acid stabilized formulation that is self-buffered. The buffer may be included in an amount sufficient to maintain the selected pH of the formulation under storage conditions for shelf stability of the product, for example, 36 months at about 2°C to about 8°C, optionally followed by about 1 month at about 20°C to about 30°C.

The aqueous pharmaceutical formulation may comprise a buffer, and optionally, the buffer is centered, at 25°C, in a range of about pH 4.0 to about pH 5.5. The buffer may have a pKa within one pH unit of pH 5.0-5.2 at 25°C. The aqueous pharmaceutical formulation may comprise about 5 mM to about 60 mM buffer, about 5 mM to about 50 mM buffer, or about 9 mM to about 45 mM buffer (e.g., about 15 mM to about 30 mM buffer, e.g., about 20 mM, about 25 mM buffer). The buffer may be acetate or glutamate.

The formulation may also include one or more protein aggregation stabilizers and other formulation excipients. Such stabilizers and excipients are contemplated to include, but are not limited to, amino acid aggregation inhibitors, tonicity modifiers, surfactants, solubilizing agents (e.g., N-methyl-2-pyrrolidone), PEG conjugation, and cyclodextrins (e.g., Captisol®).

The term "amino acid aggregation inhibitor" refers to an amino acid or a combination of amino acids (e.g., mixtures, or dipeptides, or oligopeptides having 2 to 10 residues), where any given amino acid is present in either its free base form or its salt form (e.g., arginine HCl), or an amino acid analogue, and which reduces HMWS or inhibits the formation of HMWS. Contemplated salts include sodium salts, potassium salts, and hydrochloride salts. Additionally, salts of arginine with hydrochloride, glutamate, butyrate, and glycolate are contemplated. Where a combination of amino acids is used, all of the amino acids may be present in their free base forms, all may be present in their salt forms, or some may be present in their free base forms while others are present in their salt forms. In addition to or as an alternative to dipeptides and oligopeptides, mixtures of one or more amino acids may be used, for example, a mixture of arginine and phenylalanine. Only one type of amino acid aggregation inhibitor may be present in the aqueous pharmaceutical formulation. Only one amino acid may be present, for example, only L-arginine or only L-phenylalanine may be present in the formulation.

The use of one or more amino acids bearing a charged side chain is contemplated, for example, one or more of arginine, lysine, histidine, aspartate, and glutamate. The amino acids may be selected from basic amino acids, for example, arginine, lysine, histidine, or a combination thereof. Arginine is particularly contemplated. Any stereoisomer (i.e., L-, D-, or DL-isomer) of a particular amino acid, or combinations of these stereoisomers, or formulation may be used in the present method as long as the particular amino acid is present in its free base form or its salt form. An L-stereoisomer, for example, L-arginine, is particularly contemplated. Optionally, the amino acid is one having a positively charged side chain, for example, arginine.

Optionally, one or more amino acids with aromatic rings in their side chains are contemplated, for example, phenylalanine, tyrosine, tryptophan, or a combination thereof. Phenylalanine is particularly contemplated.

Optionally, the use of one or more hydrophobic amino acids is contemplated, for example, alanine, isoleucine, leucine, phenylalanine, valine, proline or glycine.

Optionally, the use of one or more aliphatic hydrophobic amino acids, for example, alanine, isoleucine, leucine, or valine, is contemplated. Leucine is particularly contemplated.

Amino acid analogs that exhibit aggregation-reducing or -inhibiting effects may also be used in the present method or formulation. The term "amino acid analog" refers to a naturally occurring derivative of the amino acid. Contemplated analogs include, for example, amino and N-monoethyl, and N-ethyl derivatives. Other contemplated analogs include dipeptides, or oligopeptides having 2 to 10 residues, for example, arginine-arginine and phenylalanine-arginine. Optionally, it is contemplated that N-acetyl arginine and N-acetyl lysine are not used alone, but may be used in combination with another amino acid aggregation inhibitor. As with amino acids, amino acid analogs are used in the present method or formulation in either their free base form or their salt form.

The amino acid aggregation inhibitor(s) used in the present method or formulation protect the therapeutically active protein from various stresses, thereby increasing and/or maintaining the stability of the protein or formulation containing the protein over the shelf life of the protein (before and during storage, prior to use). As used herein, the term "stress" includes, but is not limited to, heat, freezing, pH, light, agitation, oxidation, dehydration, surfaces, shear, freeze/thaw, pressure, heavy metals, phenolic compounds, denaturants, etc., from any source, e.g., transportation. Heat stress is particularly contemplated. The term stress encompasses any factor that modulates (i.e., reduces, maintains, or increases) the stability of a protein or a formulation containing the protein. The increase and/or maintenance of stability with the addition of an amino acid aggregation inhibitor occurs in a concentration-dependent manner. That is, increasing concentrations of amino acid aggregation inhibitor lead to increased and/or maintained stability of a protein or a formulation containing a protein of the present disclosure when that protein or formulation containing that protein normally exhibits aggregate formation in the absence of the amino acid aggregation inhibitor. As shown in the examples below, inclusion of an amino acid aggregation inhibitor in the formulation can also reduce the amount of HMWS already formed. For example, such amino acid aggregation inhibitors include arginine and the dipeptide arginine-phenylalanine. Determining the amount of a particular amino acid aggregation inhibitor to be used in the present method or formulation to reduce aggregate formation thereby increasing protein stability, and thereby increasing the stability of the formulation over the lifetime of the protein, can be readily determined for denosumab or any particular human anti-RANKL monoclonal antibody of interest in view of the disclosure herein.

The presence of an amino acid aggregation inhibitor in the formulation has been shown to reduce the amount of dimeric species and their kinetic rate of formation. For example, including arginine at a concentration of 75 mM in a denosumab formulation having a pH of 5.2 resulted in approximately 0.3% and 25% reductions in the amounts of dimeric species and their kinetic rate of formation, respectively, after 1 month at 37°C when compared to a similar formulation without arginine at pH 5.2. In contrast, a monoclonal antibody other than a human anti-RANKL monoclonal antibody was found not to be stabilized by the inclusion of arginine and instead resulted in an increase in HMWS. Accordingly, another method of the disclosure is a method of reducing HMWS in a formulation of denosumab or other human anti-RANKL monoclonal antibody by adding an amino acid aggregation inhibitor, e.g., arginine or phenylalanine.

Accordingly, the aqueous pharmaceutical formulation may comprise an amino acid aggregation inhibitor, which optionally is an amino acid. The amino acid may be an L-stereoisomeric amino acid (L-amino acid), although D-stereoisomeric amino acids (D-amino acids) are contemplated. The amino acid aggregation inhibitor may comprise an amino acid comprising a charged side chain, also referred to herein as a "charged amino acid." The term "charged amino acid" refers to an amino acid comprising a side chain that is negatively charged (i.e., deprotonated) or negatively charged (i.e., protonated) in aqueous solution at physiological pH. For example, negatively charged amino acids include, for example, aspartic acid and glutamic acid, while positively charged amino acids include, for example, arginine, lysine, and histidine. Charged amino acids include the charged amino acids among the 20 encoded amino acids, as well as atypical or non-naturally occurring or unencoded amino acids. Accordingly, the amino acid aggregation inhibitor may be an amino acid comprising a positively charged side chain. The amino acid comprising a positively charged side chain may comprise a side chain structure of Formula I or Formula II:

wherein n is 1 to 7, wherein each of R<i>and R<2> is independently selected from the group consisting of H, C<1>-C<18>alkyl, (C<1>-C<18>alkyl)OH, (C<1>-C<18>alkyl)NH<2>, NH, NH<2>(C<1>-C<18>alkyl)SH, (C<0>-C<4>alkyl)(C<3>-C<6>cycloalkyl), (C<0>-C<4>alkyl)(C<2>-C<5>heterocyclic), (C<0>-C<4>alkyl)(C<6>-C<10>aryl)R<7>and (C<1>-C<4>alkyl)(C<3>-C<9>heteroaryl), wherein R<7>is H or OH, wherein optionally one of R<1>and R<2>is a free amino group (-NH<3+>),

[Formula II]

wherein m is 1 to 7, wherein each of R<3> and R<4> is independently selected from Group A consisting of: H, C<1>-C<18> alkyl, (C<1>-C<18> alkyl)OH, (C<1>-C<18> alkyl)NH<2>, (C<1>-C<18> alkyl)SH, (C<0>-C<4> alkyl)(C<3>-C<6> cycloalkyl), (C<0>-C<4> alkyl)(C<2>-C<5> heterocyclic), (C<0>-C<4> alkyl)(C<6>-C<10> aryl)R<8> and (C<1>-C<4> alkyl)(C<3>-C<9> heteroaryl), wherein R<8> is H or OH, wherein, R<5> is optionally present, and, when present, is selected from the group A, optionally, where each of R<3>and R<4>and R<5>is H.

The amino acid comprising a positively charged side chain may comprise a side chain structure of formula I and, n is in a range of 2 to 4. R<1> may be NH or NH<2>. R<2> may be NH<2> or NH<3+>. The amino acid comprising a positively charged side chain may be arginine. The amino acid comprising a positively charged side chain may comprise a side chain structure of formula II and, m is in a range of 3 to 5. Each of R<3> and R<4> may be H. R<5> may be present, and optionally is H. The amino acid comprising a positively charged side chain may be lysine. The amino acid comprising a positively charged side chain is present in the formulation as a salt, optionally, a hydrochloride (HCl) salt. Accordingly, the aqueous pharmaceutical composition may comprise L-arginine HCl or L-lysine HCl.

The amino acid aggregation inhibitor may be an aromatic amino acid. The aromatic amino acid may comprise a phenyl or an indole. The aromatic amino acid may comprise a C<1>-C<6> alkyl chain (e.g., a C<1>-C<3> alkyl chain) between the alpha carbon and the phenyl or indole. The aromatic amino acid may be L-phenylalanine. The aromatic amino acid may be L-tryptophan.

The amino acid aggregation inhibitor may be a hydrophobic amino acid. Hydrophobicity may be measured or scored according to any one of the hydrophobicity scales known in the art. In general, the more positive the score, the more hydrophobic the amino acid. Hydrophobicity may be scored on the Kyte and Doolittle hydrophobicity scale (Kyte J, Doolittle RF (May 1982). "A simple method for displaying the hydropathic character of a protein". J. Mol. Biol. 157 (1): 105-32.) The hydrophobic amino acid may have a score greater than about 2.5 on the Kyte and Doolittle hydrophobicity scale. The hydrophobic amino acid may comprise a side chain comprising a C<2> to C<12> alkyl, branched or straight chain, or a C<4> to C<8> cycloalkyl, a C<4> to C<8> heterocycle comprising a nitrogen heteroatom, optionally, wherein the heterocycle is an imidazole, pyrrole, or indole. For purposes herein, the term "cycloalkyl" encompasses any carbon cycle, including carbon bicycles or tricycles.

The hydrophobic amino acid may comprise a C<3> to C8 alkyl, optionally, the hydrophobic amino acid comprises a branched C<3> alkyl or a branched C<4> alkyl. The hydrophobic amino acid may be L-valine, L-leucine or L-isoleucine.

The amino acid aggregation inhibitor is used in an amount effective to provide high stability, and can be used in a concentration in a range of about 10 mM to about 200 mM, for example, a range of about 30 mM to about 120 mM, or about 38 mM to about 150 mM, or about 38 mM to about 113 mM, or about 38 mM to about 75 mM, for example about 10 mM, about 38 mM, about 75 mM, about 113 mM or about 150 mM. The aqueous pharmaceutical formulation can comprise about 5 mM to about 300 mM amino acid aggregation inhibitor, optionally, about 25 mM to about 90 mM amino acid aggregation inhibitor. The aqueous pharmaceutical formulation may comprise about 5 mM to about 150 mM amino acid aggregation inhibitor (e.g., about 10 mM to about 150 mM, about 15 mM to about 150 mM, about 20 mM to about 150 mM, about 25 mM to about 150 mM, about 5 mM to about 140 mM, about 5 mM to about 130 mM, about 5 mM to about 120 mM, about 5 mM to about 110 mM, about 5 mM to about 100 mM, about 5 mM to about 90 mM), when the amino acid aggregation inhibitor is an amino acid comprising a positively charged side chain, optionally, L-arginine. The aqueous pharmaceutical formulation may comprise about 30 mM to about 80 mM amino acid aggregation inhibitor (e.g., about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM), when the amino acid aggregation inhibitor is an amino acid comprising a positively charged side chain, optionally, L-arginine.

The aqueous pharmaceutical formulation may comprise about 5 mM to about 180 mM amino acid aggregation inhibitor (e.g., about 10 mM to about 180 mM, about 15 mM to about 180 mM, about 20 mM to about 180 mM, about 25 mM to about 180 mM, about 5 mM to about 170 mM, about 5 mM to about 170 mM, about 5 mM to about 160 mM, about 1 mM to about 150 mM, about 5 mM to about 140 mM, about 5 mM to about 130 mM, about 5 mM to about 120 mM, about 5 mM to about 110 mM), when the amino acid aggregation inhibitor is an aromatic amino acid, optionally, L-phenylalanine. The aqueous pharmaceutical formulation may comprise about 5 mM to about 100 mM amino acid aggregation inhibitor (e.g., about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM), optionally, about 20 mM to about 50 mM amino acid aggregation inhibitor, when the amino acid aggregation inhibitor is an aromatic amino acid, optionally, L-phenylalanine.

Optionally, the aqueous pharmaceutical formulation comprises about 5 mM to about 300 mM amino acid aggregation inhibitor, when the amino acid aggregation inhibitor is a hydrophobic amino acid, optionally, L-valine, L-isoleucine or L-leucine. Optionally, the aqueous pharmaceutical formulation comprises about 5 mM to about 200 mM amino acid aggregation inhibitor (e.g., about 10 mM to about 200 mM, about 20 mM to about 200 mM, about 30 mM to about 200 mM, about 40 mM to about 200 mM, about 50 mM to about 200 mM, about 60 mM to about 200 mM, about 70 mM to about 200 mM, about 80 mM to about 200 mM, about 90 mM to about 200 mM, about 100 mM to about 200 mM, about 5 mM to about 290 mM, about 5 mM to about 280 mM, about 5 mM to about 270 mM, about 5 mM to about 260 mM, about 100 mM to about 200 mM). 5 mM to about 250 mM, about 5 mM to about 240 mM, about 5 mM to about 230 mM, about 5 mM to about 220 mM, about 5 mM to about 210 mM), optionally, about 20 mM to about 50 mM amino acid aggregation inhibitor, when the amino acid aggregation inhibitor is a hydrophobic amino acid, optionally, L-valine, L-isoleucine or L-leucine. The aqueous pharmaceutical composition may comprise: about 30 mM to about 80 mM L-arginine hydrochloride; about 20 mM to about 50 mM L-phenylalanine; about 20 mM to about 50 mM L-tryptophan; about 30 mM to about 80 mM L-lysine hydrochloride; L-leucine from about 20 mM to about 50 mM; L-isoleucine from about 20 mM to about 50 mM; L-valine from about 20 mM to about 50 mM; or any combination thereof.

The concentration of the amino acid aggregation inhibitor may be in molar ratio to the antibody. The molar ratio of the amino acid aggregation inhibitor to the anti-RANKL antibody may be about 10 to about 200 (e.g., about 25 to about 150, about 50 to about 100), when the amino acid aggregation inhibitor is an aromatic amino acid, optionally, L-phenylalanine. Optionally, the molar ratio is about 20 to about 90. The molar ratio of the amino acid aggregation inhibitor to the anti-RANKL antibody may be about 20 to 300, when the amino acid aggregation inhibitor is an amino acid comprising a positively charged side chain, optionally, L-arginine. Optionally, the molar ratio is about 45 to about 180.

Surfactants are surface-active agents that are amphipathic (having a polar head and a hydrophobic tail). Surfactants accumulate preferentially at interfaces, resulting in reduced interfacial tension. A surfactant may be optionally included in the formulation. The use of a surfactant can also help mitigate the formation of large proteinaceous particles.

The surfactant may be a non-ionic surfactant. Examples include polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 80), alkylaryl polyethers, e.g., oxyethylated alkyl phenols (e.g., Triton™ X-100), and poloxamers (e.g., Pluronics®, e.g., Pluronic® F68), and combinations of any of the foregoing, either within a surfactant class or between surfactant classes. Particularly contemplated are polysorbate 20 and polysorbate 80.

A surfactant concentration in a range of about 0.004% (w/v) to about 0.1% (w/v) (e.g., for polysorbate 20 or polysorbate 80) is suitable, e.g., about 0.004% to about 0.05%, or about 0.004% to about 0.02%, or about 0.01%. The formulation may comprise at least about 0.004% (w/v) of surfactant, and optionally, less than about 0.15% (w/v). About 0.005% (w/v) to about 0.015% (w/v) of surfactant may be present in the formulation, optionally, about 0.005% (w/v), about 0.006% (w/v), about 0.007% (w/v), about 0.008% (w/v), about 0.009% (w/v), about 0.010% (w/v), about 0.011% (w/v), about 0.012% (w/v), about 0.013% (w/v), or about 0.014% (w/v).

The stabilized aqueous formulation may be suitable for administration by any acceptable route, including parenteral and specifically subcutaneous. For example, subcutaneous administration may be to the upper arm, upper thigh, or abdomen. Other routes include, for example, intravenous, intradermal, intramuscular, intraperitoneal, intranodal, and intrasplenic. The subcutaneous route is preferred.

51 The solution is in a form intended for administration to a subject, it can be prepared to be isotonic with the intended administration site. For example, the osmolality can be in a range of about 270 to about 350 mOsm/kG, or about 285 to about 345 mOsm/kG, or about 300 to about 315 mOsm/kG. For example, if the solution is in a form intended for parenteral administration, it can be isotonic with blood (osmolality of about 300 mOsm/kG). The aqueous pharmaceutical formulation can have an osmolality in a range of about 200 mOsm/kg to about 500 mOsm/kg, or about 225 mOsm/kg to about 400 mOsm/kg, or about 250 mOsm/kg to about 350 mOsm/kg.

The aqueous pharmaceutical formulation may have a conductivity in a range of about 500 pS/cm to about 5500 pS/cm, optionally, wherein the conductivity is in a range of about 2500 pS/cm to about 5500 pS/cm, when the formulation comprises an amino acid comprising a positively charged side chain, or in a range of about 500 pS/cm to about 2000 pS/cm, when the formulation comprises an aromatic amino acid or lacks the amino acid aggregation inhibitor. The aqueous pharmaceutical formulation may have a viscosity that is no more than about 6 cP at 5 ° C, optionally, wherein the viscosity is about 4.5 cP to about 5.5 cP. The aqueous pharmaceutical formulation may have a viscosity that is less than about 13 cP at 25 ° C, optionally, about 2.0 cP to about 10 cP, optionally, about 2.5 cP to about 4 cP.

Tonicity modifiers, or tonicity adjusting agents, are known in the art, and include compounds such as salts (e.g., sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium bicarbonate, calcium carbonate, sodium lactate), sugars (e.g., dextran, dextrose, lactose, trehalose), and sugar alcohols (e.g., mannitol, sorbitol, xylitol, glycerol, propylene glycol). The tonicity modifier may be selected from the group consisting of: sorbitol, mannitol, sucrose, trehalose, glycerol, and combinations thereof. The tonicity modifier may be sorbitol. Sorbitol may be used, for example, in a concentration in a range of 0.1% (w/v) to 5% (w/v), or 1.2% (w/v) to 5% (w/v), for example 3.6% (w/v), 4.6% (w/v), or 4.7% (w/v). Optionally, the formulation comprises about 1.0% (w/w) to about 5.0% (w/w) of tonicity modifier. For example, the formulation comprises about 2.0% (w/w) to about 5.0% (w/w) sorbitol, or about 3.5% (w/w) to about 5.0% (w/w) sorbitol, or about 4.0% (w/w) to about 5.0% (w/w) sorbitol. Optionally, the formulation does not comprise sorbitol or is sorbitol-free. Optionally, the formulation does not include a tonicity modifier.

Other excipients known in the art can be used in the formulation, as long as they do not adversely affect stability. Sugars and polyols can be used to protect proteins from aggregation, including providing freeze/thaw stability. Such compounds include sorbitol, mannitol, glycerol, erythritol, caprylate, tryptophanate, sarcoside, and glycine. Stabilizers can also be used to prepare lyophilized preparations, for example, stabilizing sugars, for example, disaccharides such as trehalose and sucrose. A lyophilized preparation can also include a thickener, as is known in the art. Other excipients known in the art for stabilizing proteins include solubilizing agents (e.g., N-methyl-2-pyrrolidone), polyethylene glycol (PEG), and cyclodextrins (e.g., Captisol®). Pharmaceutically acceptable acids and bases may be used to adjust the pH of the solution, e.g., sodium hydroxide.

For parenteral administration, the formulation may be in the form of a sterile, parenterally acceptable, pyrogen-free aqueous solution comprising denosumab or another human anti-RANKL monoclonal antibody, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle. A vehicle for parenteral injection may be sterile distilled water in which the denosumab or other human anti-RANKL monoclonal antibody, with or without at least one additional therapeutic agent, is formulated as a sterile, isotonic solution. The formulation will contain pharmaceutically acceptable excipients, e.g., USP (United States Pharmacopeia) grade excipients.

A "preservative" is a compound that can be included in a pharmaceutical formulation to reduce bacterial action on it, for example, thereby facilitating the production of a multi-use formulation. Examples of preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols including phenol, butyl and benzyl alcohol, alkyl parabens including methyl and propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and mcresol. Alternatively, the formulation may be preservative-free. For example, the formulation, when presented in a single-use dosage form, may be preservative-free.

Although the formulation has been described herein in its aqueous form, the stabilized formulation can also be subsequently lyophilized to prepare a lyophilizate. Accordingly, unless the context clearly dictates otherwise, references to the formulation and its method are intended to include a lyophilizate resulting from the stabilized aqueous solution.

The pharmaceutical formulation to be used for in vivo administration is normally sterile. This can be accomplished by filtration through sterile filtration membranes. The parent compositions can generally be placed in a container having a sterile access port, for example, an intravenous dissolution bag, or a vial having a stopper pierceable by a hypodermic injection needle, or a prefilled syringe. The formulation can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted or diluted prior to administration.

Described herein are kits for producing a single-dose dosage unit. Each kit may contain both a first container containing a dried preparation of denosumab or another human anti-RANKL monoclonal antibody prepared from a solution formulation described herein, and a second container containing sterile water or an aqueous solution. The kits may contain prefilled single- and multi-chamber syringes (e.g., liquid syringes and lyo-syringes).

The stabilized formulation described herein may be used in conjunction with one or more additional therapeutic agents, for example calcium and a vitamin D compound. The stabilized formulation described herein may be administered to a patient receiving therapy with an additional therapeutic agent, or the stabilized formulation described herein may be co-administered with an additional therapeutic agent.

The stabilized formulation can be used to prevent or treat any disease responsive to denosumab or another human anti-RANKL monoclonal antibody, or an antigen-binding portion thereof. Such uses and related methods include, but are not limited to, the disclosures described below.

The formulation may be used to prevent a skeletal-related event (SRE) in a patient in need thereof, including administering an effective amount of a stabilized formulation described herein. The SRE may be selected, for example, from the group consisting of a pathological fracture, radiation therapy to bone, surgery to bone, and spinal cord compression. The patient may be one having bone metastasis from a solid tumor. The solid tumor may be, for example, one or more of breast cancer, prostate cancer, lung cancer, non-small cell lung cancer, and renal cell carcinoma. The amount of the formulation may be effective to reduce the creatinine-corrected urinary amino-terminal telopeptide of bone turnover marker (uNTx/Cr), optionally by at least 80%. The patient may be a patient with multiple myeloma.

The formulation can be used to treat a patient with a giant cell tumor of bone, including administering an effective amount of a stabilized formulation described herein. The patient may have a giant cell tumor of bone that is recurrent, inoperable, or for which surgical resection is likely to result in severe morbidity. The patient may be, for example, a skeletally mature adult or adolescent.

The formulation may be used to treat a patient with hypercalcemia of a malignant bone tumor, including administering an effective amount of a stabilized formulation described herein. The malignant tumor may be resistant to bisphosphonate therapy. The method or use may include administering an amount of the formulation effective to reduce or maintain the patient's serum calcium to a level less than or equal to about 11.5 mg/dL.

The formulation can be used to treat osteoporosis in a patient in need thereof, including administering an effective amount of a stabilized formulation described herein. For example, the patient may be a postmenopausal woman at high risk of fracture. The patient may be a man at high risk of fracture.

The formulation can be used to increase bone mass in a patient in need thereof, which includes administering an effective amount of a stabilized formulation described herein. For example, the amount of formulation administered can be an amount effective to reduce the incidence of new vertebral fractures and/or non-vertebral fractures. The amount of formulation administered can be an amount effective to reduce bone resorption. The amount of formulation can be an amount effective to increase bone density in the patient in at least a selected area of the lumbar spine, the entire hip, and the femoral neck. The amount of formulation can be an amount effective to increase bone mass in the patient's cortical bone and/or trabecular bone. The amount of formulation can be an amount effective to reduce the bone resorption marker serum C-telopeptide type 1 (CTX). The patient in need thereof can optionally have osteoporosis. The patient in need thereof can be a woman at high risk of fracture receiving adjuvant aromatase inhibitor therapy for breast cancer. The patient in need of this treatment may be a man at high risk for fracture who is receiving androgen deprivation therapy for non-metastatic prostate cancer. The patient in need of this treatment may be a man with osteoporosis who is at high risk for fracture.

The formulation can be used as an adjuvant treatment for postmenopausal women with early-stage breast cancer at high risk of disease recurrence who are receiving adjuvant/neoadjuvant cancer therapy.

The formulation can be used as a first-line treatment for patients with metastatic non-small cell lung cancer in combination with platinum-based chemotherapy.

The formulation can be used to treat idiopathic subglottic stenosis (ISS).

The formulation can be used for the prevention of breast and ovarian cancer in healthy women with BRCA-1 mutations.

Optionally, the formulation can be used in combination with an immune checkpoint inhibitor. Optionally, the immune checkpoint inhibitor is specific for a protein that functions in an immune checkpoint pathway, for example, for example, CTLA4, LAG3, PD-1, PD-L1, PD-L2, B7-H3, B7H4, BTLA, SLAM, 2B4, CD160, KLRG-1, or TIM3. Optionally, the immune checkpoint inhibitor is an antibody, antigen-binding fragment thereof, or an antibody protein product specific for CTLA4, LAG3, PD-1, PD-L1, PD-L2, B7-H3, B7H4, BTLA, SLAM, 2B4, CD160, KLRG-1, or TIM3. Such immune checkpoint inhibitors include, but are not limited to: atezolizumab, avelumab, ipilimumab, tremelimumab, BMS-936558, MK3475, CT-011, AM-224, m Dx -1105, IMP321, MGA271. PD-1 inhibitors include, for example, pembrolizumab and nivolumab. PD-L1 inhibitors include, for example, atezolizumab, avelumab, and durvalumab. CTLA4 inhibitors include, for example, ipilimumab. The formulation may be used to treat patients with melanoma with bone metastases, optionally in combination with a PD-1 antibody (e.g., nivolumab, pembrolizumab). The formulation may be used to treat patients with breast cancer, optionally, in combination with a CTLA4 inhibitor, such as ipilimumab.

The formulation can be used to treat tumors rich in giant cells, for example, in hyperparathyroidism or with a secondary aneurysmal bone cyst.

The formulation can be used to treat progressive metastatic castration-resistant prostate cancer (mCRPC). The formulation can be used to treat castration-sensitive prostate cancer. The formulation can be used to treat hormone-resistant prostate cancer.

The formulation can be used to treat metastatic breast cancer (mBC). The formulation can be used to treat preoperative breast cancer. The formulation can be used to treat early breast cancer. The formulation can be used to treat hormone receptor-negative, RANK-positive, or RANK-negative primary breast cancer. The formulation can be used to treat postmenopausal HER2-negative breast cancer.

The formulation can be used to treat myelodysplastic syndrome, for example, in an elderly patient.

The formulation can be used to treat cancer treatment-induced bone loss (CTIBL).

The formulation can be used to treat a uterine tumor of the cervix.

The formulation can be used to induce immunomodulatory effects in patients with or without immunotherapy.

The formulation can be used to prevent or treat bone loss associated with osteoporosis, Paget's disease, osteomyelitis, hypercalcemia, osteopenia, osteonecrosis, and rheumatoid arthritis. The formulation can be used to prevent or treat inflammatory conditions with bone loss. The formulation can be used to prevent or treat autoimmune conditions with bone loss. The formulation can be used to prevent or treat bone loss associated with cancer, including breast, prostate, thyroid, kidney, lung, esophageal, rectal, bladder, cervical, ovarian, liver, and gastrointestinal cancers, multiple myeloma, lymphoma, and Hodgkin's disease.

The formulation can be administered on any suitable chronological schedule. The dosing schedule may be once every four weeks. Optionally, dosing may include administration on days 8 and 15 of the first month of therapy. Dosing may be on a once-every-six-month schedule. For example, a once-every-six-month schedule is contemplated for use with osteoporosis and increasing bone mass. Other maintenance dosages contemplated are every 3 weeks, every 3 months, and every 6 weeks.

The aqueous pharmaceutical formulation can be used to treat a patient with multiple myeloma or bone metastasis from a solid tumor. The formulation can be administered in doses of approximately 120 mg every 4 weeks as a subcutaneous injection in the upper arm, upper thigh, or abdomen.

The aqueous pharmaceutical formulation can be used to treat a patient with a giant cell tumor of bone. The formulation can be administered in doses of approximately 120 mg every 4 weeks, with additional doses of 120 mg on days 8 and 15 of the first month of therapy. The formulation can be administered subcutaneously in the patient's upper arm, upper thigh, or abdomen. Calcium and vitamin D can be administered to the patient to treat or prevent hypocalcemia.

The aqueous pharmaceutical formulation can be used to treat a patient with hypercalcemia of malignancy. The formulation can be administered in doses of approximately 120 mg every 4 weeks, with additional doses of 120 mg on days 8 and 15 of the first month of therapy. The formulation can be administered subcutaneously in the upper arm, upper thigh, or abdomen.

The aqueous pharmaceutical formulation can be used to treat postmenopausal women with osteoporosis at high risk of fracture, or to increase bone mass in men at high risk of fracture receiving androgen deprivation therapy for non-metastatic prostate cancer, or in women at high risk of fracture receiving adjuvant aromatase inhibitor therapy for breast cancer. The aqueous pharmaceutical formulation can be administered by a healthcare professional in doses of 60 mg every 6 months as a subcutaneous injection in the upper arm, upper thigh, or abdomen. The patient may be instructed to take 1000 mg of calcium daily and at least 400 IU of vitamin D daily.

One type of formulation according to the disclosure will contain denosumab, acetate, and arginine. The arginine is optionally L-arginine. The arginine is optionally L-arginine hydrochloride. The formulation may optionally include sorbitol. The formulation may optionally include polysorbate. The polysorbate may optionally be polysorbate 20. The pH may optionally be about 5.0 to about 5.2, or less than 5.2.

Another type of formulation according to the disclosure will contain denosumab, acetate, and phenylalanine. The formulation may optionally include sorbitol. The formulation may optionally include polysorbate. The polysorbate may optionally be polysorbate 20. The pH may optionally be about 5.0 to about 5.2, or less than 5.2. For example, the formulation may include denosumab at a concentration of about 108 mg/ml to about 132 mg/ml, acetate of about 28.8 mM to about 35.2 mM, phenylalanine of 33.3 mM to about 40.7 mM, 3.51% (w/v) to about 4.29% (w/v) sorbitol, and about 0.009% (w/v) to about 0.011% (w/v) polysorbate 20, a pH 5.1, and may optionally be contained in a PFS, optionally containing about 1 ml or less than about 1 ml (e.g., about 0.5 ml) of formulation. For example, the formulation may include denosumab at a concentration of 120 mg/ml, 32 mM acetate, 37 mM phenylalanine, 3.9% (w/v) sorbitol, and 0.01% (w/v) polysorbate 20, a pH of 5.1, and may optionally be contained in PFS, optionally containing about 1 ml or less than about 1 ml (e.g., about 0.5 ml) of formulation. The formulation may be prepared by concentrating denosumab using a diafiltration buffer containing 20 mM acetate, 4.2% (w/v) sorbitol, and 40 mM phenylalanine, a pH of 4.7.

Another type of formulation according to the disclosure will contain denosumab, glutamate, and arginine. The arginine is optionally L-arginine. The arginine is optionally L-arginine hydrochloride. The formulation may optionally include sorbitol. The formulation may optionally include polysorbate. The polysorbate may optionally be polysorbate 20. The pH may optionally be about 5.0 to about 5.2, or less than 5.2.

Another type of formulation according to the disclosure will contain denosumab, acetate, arginine, and phenylalanine. The formulation may optionally include sorbitol. The formulation may optionally include polysorbate. The polysorbate may optionally be polysorbate 20. The pH may optionally be about 5.0 to about 5.2, or less than 5.2.

Another type of formulation according to the disclosure will contain denosumab, glutamate, arginine, and phenylalanine. The arginine is optionally L-arginine. The arginine is optionally L-arginine hydrochloride. The formulation may optionally include sorbitol. The formulation may optionally include polysorbate. The polysorbate may optionally be polysorbate 20. The pH may optionally be about 5.0 to about 5.2, or less than 5.2.

The formulations according to the disclosure can be prepared by any suitable method. In one type of method, a solution containing a monoclonal anti-RANKL antibody (e.g., denosumab) can be prepared at a concentration below 70 mg/ml, a suitable amount of the amino acid aggregation inhibitor described herein can be added to the solution, and then the solution can be concentrated to an amount greater than 70 mg/ml described herein, e.g., 120 mg/ml. Optionally, the solution can first be superconcentrated, i.e., to a concentration of monoclonal anti-RANKL antibody (e.g., denosumab) greater than the final target concentration, and then the superconcentrated solution can be diluted, e.g., with a pH-adjusted buffer solution, to the final target concentration and pH. For example, the overconcentration may result in an amount of anti-RANKL monoclonal antibody (e.g., denosumab) in a range of 130 mg/ml to 300 mg/ml or 180 mg/ml to 300 mg/ml. The initial concentration of denosumab prior to concentration is not particularly limited, and may be, for example, about 1 mg/ml, or about 2 mg/ml, or about 5 mg/ml, or about 8 mg/ml, or about 10 mg/ml, or about 20 mg/ml, or about 30 mg/ml, or about 40 mg/ml, or about 50 mg/ml, or about 60 mg/ml, or about 70 mg/ml, or in a range encompassed by any of such concentrations, for example, about 1 mg/ml to about 70 mg/ml, or about 1 mg/ml to about 10 mg/ml.

The formulation can be concentrated by any suitable method. The concentration process may include centrifugation. The concentration process may include ultrafiltration.

The introduction of the amino acid aggregation inhibitor into the formulation can be done by any suitable process. For example, the amino acid aggregation inhibitor can be introduced into the formulation by a simple addition (spiking) to the formulation, for example, as described in the examples below. In another method, the amino acid aggregation inhibitor can be introduced into the formulation by diafiltration against a buffer solution containing the amino acid aggregation inhibitor, for example, as described in the examples below. The amino acid aggregation inhibitor can be introduced into the formulation before or after concentrating the anti-RANKL monoclonal antibody above 70 mg/ml. As shown in the examples below, there is a benefit to adding the amino acid aggregation inhibitor to the solution before concentration, as it inhibits aggregation during the concentration process.

Accordingly, the disclosure provides methods of preparing a stable aqueous pharmaceutical formulation, comprising an anti-human receptor activator of nuclear factor kappa-B ligand (anti-RANKL) human monoclonal antibody, or an antigen-binding portion thereof. The method may comprise combining the anti-RANKL monoclonal antibody, or antigen-binding portion thereof, at a concentration greater than 70 mg/ml with an amino acid aggregation inhibitor, a buffer, a surfactant, and optionally, a tonicity modifier. The antibody, or antigen-binding portion, may be any of those described herein, and the concentration of the antibody, or antigen-binding portion thereof, may be according to the teachings herein. The amino acid aggregation inhibitor may be any of those described herein. For example, the amino acid aggregation inhibitor may be a positively charged amino acid, an aromatic amino acid, or a hydrophobic amino acid. The amino acid aggregation inhibitor can be in molar ratio to the antibody as described herein. The amount and selection of aggregation inhibitor, surfactant, tonicity modifier, and buffer are as described above. The disclosure also provides formulations made by the preparation methods described herein.

A formulation according to the disclosure herein may include adjusting the pH of a high concentration monoclonal anti-RANKL antibody solution (e.g., denosumab) described herein, for example, one having a concentration greater than 70 mg/ml, or 120 mg/ml. The formulation may be made by adjusting the pH of a low concentration monoclonal anti-RANKL antibody solution (e.g., denosumab) and then concentrating the solution to the highest desired concentration. Suitable pH adjusting agents are known in the art.

EXAMPLES

The following examples are provided for illustration and are not intended to limit the scope of the invention. Throughout the examples presented herein, the following abbreviations are used: DF, diafiltration; PS20, polysorbate 20; HCl, hydrochloride; UF/DF, ultrafiltration/diafiltration; F#, formulation number; HMWS, high molecular weight species; SE-UHPLC, size exclusion ultra-high performance liquid chromatography. Furthermore, in all of these examples, the composition of the DF buffer or dialysis buffer is used to make the final formulation comprising denosumab, and estimated concentrations of the components of the final formulation are provided. The final concentrations of certain components of the final formulations stored and subsequently tested for stability can be differentiated from the concentrations of DF or dialysis buffer depending on the presence or absence of a counterion (e.g., HCl). Without a counterion, the formulations have low ionic strength. In such cases, acetate is co-concentrated with denosumab such that the final formulations comprise a higher concentration of acetate, relative to the concentration of DF or dialysis buffer. For example, use of a DF buffer comprising 10 mM acetate results in ~23 mM acetate in the final denosumab formulation (120 mg/ml) (pH 5.1), when neither the DF buffer nor the final formulation comprises a counterion (e.g., HCl) and is thus of low ionic strength. Similarly, a DF buffer comprising 20 mM acetate results in ~32 mM acetate in the final denosumab formulation (120 mg/ml) at pH 5.1, without a counterion (e.g., HCl). When a counterion (e.g., arginine HCl) is present, acetate does not co-concentrate with denosumab, and therefore, the acetate concentration of the DF buffer and the acetate concentration of the final composition are generally equivalent. Furthermore, excipients may be volumetrically excluded or may be affected by non-specific interactions. For example, in a 120 mg/mL denosumab formulation, the concentrations of phenylalanine and sorbitol are approximately 7-10% lower than those stated in the DF buffer, and the arginine concentration is approximately 10-15% lower. In view of the foregoing, all of the following exemplary concentrations provide the components of the final formulations, taking into account the above-described excipient exclusion and the co-concentration effects of acetate.

REFERENCE EXAMPLE 1

An initial evaluation of twelve formulations was prepared for their effect in minimizing the amount (%) of HMWS in a high concentration (120 mg/ml) denosumab liquid formulation, and its formation over time. Formulation alternatives included changes in buffer type, stabilizers, and solution pH. The formulations tested, A-L, are described in Table 1 below. All buffer values cited are for the buffer concentration against which the antibody was diafiltered. Each excipient and surfactant was added to the solution after buffer exchange at the level indicated in the table. Although acetate concentrations were not measured in the present formulations, 120 mg/ml denosumab formulations diafiltered with sorbitol against 10 mM acetate had approximate final acetate values of 25 mM to 35 mM.

Denosumab at 70 mg/ml in acetate, pH 5.2 was UF/DF against 10 mM acetate, pH 5.2 and concentrated to 160 mg/ml. Stock solutions were prepared in 10 mM acetate at pH 5.2 consisting of:

35% sorbitol

1% polysorbate 20

1% polysorbate 80

30% Pluronic® F-68

3% Triton™ X-100

L-arginine HCl 250 mM

N-acetyl-arginine (NAR) 250 mM

N-acetyl-lysine (NAK) 250 mM

Proline 250 mM

Polyethylene glycol (PEG) 3350250 mM

Captisol® Cyclodextrin 250 mM

To achieve formulations A through J, the 160 mg/ml material prepared with 10 mM acetate, pH 5.2, was diluted to 120 mg/ml using 10 mM acetate at pH 5.2, followed by an addition of the corresponding sorbitol, excipient, and/or surfactant stock solutions to a target final concentration listed in Table 1. To achieve formulations K and L, the self-buffered and glutamate formulations, respectively, two separate aliquots of the 160 mg/ml material were subjected to further buffer exchange by centrifugation. The material for formulations K and L was then diluted to 120 mg/ml using the respective buffer, followed by an addition of the corresponding sorbitol, and polysorbate 20 stock solutions to a target final concentration listed in the formulation table in Table 1.

TABLE 1

Figure 1 shows the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37°C. Formulation L, consisting of approximately 10 mM glutamate buffer, 10 mM L-arginine HCl, 2.4% (w/v) sorbitol as a tonicity modifier, and 0.01% (w/v) polysorbate 20 as a surfactant, and at a pH of 5.0, showed both reduced initial amounts of HMWS, indicating some reduction in already formed aggregates, and reduced kinetics of HMWS formation at 37°C.

REFERENCE EXAMPLE 2

Evaluation of excipient formulations of 10 mM acetate, 75 mM L-arginine, 2.4% (w/v) sorbitol, 0.01% (w/v) polysorbate 20, and an excipient formulation of 10 mM acetate, 5% (w/v) sorbitol, 0.01% (w/v) polysorbate 20, each containing high concentration denosumab (120 mg/mL), at 37°C for up to 1 month revealed the effects of pH and amino acid aggregation inhibitor on the rate and extent of HMWS formation. The formulations tested are described in Table 2 below. All buffer and excipient values quoted are for the buffer and excipient concentrations against which the antibody is diafiltered.

To prepare M-Q test samples, a 3 mL aliquot of 70 mg/mL denosumab in acetate, pH 5.2, was dialyzed against 500 mL of the DF buffer described below, with a total of 3 buffer changes to achieve a 1 million-fold dilution of the pre-formulation to ensure complete buffer exchange. The material was then superconcentrated using a centrifuge-concentrator, followed by dilution to 120 mg/mL and the addition of polysorbate 20 to a final concentration of 0.01%.

TABLE 2

Figure 2 shows the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37°C. Figure 3 shows the size exclusion chromatograms as a function of formulation after storage at 37°C for 1 month.

As the solution pH decreased, there was an increase in the formation of large aggregates. Below pH 4.8, and especially 4.5, large aggregates were the dominant HWMS, with a dramatic increase for the test formulation at pH 4.5. As shown in FIGURE 3, formulations P and Q had the lowest amount of higher-order HWMS (retention time of approximately 6 minutes), followed by comparative formulations O, N, and M having decreasing pH values.

However, as pH increased, there was, in general, a resulting increase in dimeric species. As shown in FIGURE 3, formulation N had the lowest amount of dimeric species (retention time approximately 6.8 minutes), followed by formulations M, O, P, and Q.

The presence of arginine in formulation O at a concentration of 75 mM produced approximately 0.3% and 25% reductions in the amounts of the dimeric species and their kinetic rate of formation, respectively, after 1 month at 37 °C when compared to formulation P having the same pH, but without arginine.

REFERENCE EXAMPLE 3

This example demonstrates the effect of pH on high-concentration denosumab formulations.

Denosumab (at a concentration of 120 mg/ml) was formulated with acetate, sorbitol, and polysorbate 20 (PS20) with or without an amino acid aggregation inhibitor at three different pH values: 4.8, 5.1, and 5.4. In this study, the amino acid aggregation inhibitor was L-arginine HCl. All formulations were prepared by buffer exchange of a stock solution containing a lower concentration of denosumab, followed by superconcentration of the denosumab stock and then dilution of the denosumab stock with the desired amounts of buffer, excipients, and surfactants. Briefly, an aliquot of denosumab at 70 mg/ml in acetate, pH 5.2 (starting material) was dialyzed against DF buffer as described in TABLE 3A, with a total of 3 buffer changes to achieve a 1 million-fold dilution of the starting material to ensure complete buffer exchange. The buffer-exchanged denosumab material was then concentrated using a centrifuge-concentrator to a denosumab concentration greater than 120 mg/ml, and the concentrated material was further diluted to achieve a denosumab concentration of 120 mg/ml. PS20 was added to a final concentration of 0.01%.

The protein at high concentration was believed to contribute to a solution pH based on its charge state. The acetate concentration of formulation 1 was increased to achieve the target final pH, and the acetate concentrations of formulations 2 and 3 were matched to that of formulation 1. The acetate concentrations of formulations 4-6 required an even higher amount of acetate to match the final acetate concentrations of formulations 1-3 because acetate does not co-concentrate in the presence of the HCl salt. Formulation 7 served as a control to ensure that the increased acetate concentration in formulations 4-6 did not impair protein stability in the arginine hydrochloride formulations.

The different denosumab formulations prepared and tested in this study are described in TABLE 3A.

TABLE 3A

A sample of each formulation was filled into a container with a fill volume of 1 ml and stored at 37°C for up to 4 weeks. Aggregation inhibition and stability against aggregation inhibition over time, based on the formation of HMWS and dimeric species, were evaluated using SE-UHPLC. The aggregation inhibition profiles of these formulations were compared under initial conditions and during and after the storage period.

The percentage of HMWS was monitored by SE-UHPLC as a function of formulation and time at 37 °C. FIGURE 4 represents a graph of the percentage of HMWS as a function of time for formulations 1-7 and TABLE 3B provides the data points for the graph.

TABLE 3B

Figure 5 shows the size exclusion chromatograms for each formulation after storage at 37°C for 1 month. Formulations without arginine are shown in the left panel, while formulations with arginine are shown in the right panel.

As shown in FIGURE 4, formulations containing arginine performed better than control formulations without arginine hydrochloride, and formulations at pH 5.1 performed better than comparable formulations at pH 4.8 and pH 5.4. For formulations 1-3 without arginine hydrochloride, dimeric species increased as the solution pH increased up to 5.4 (FIGURE 5A). For formulations 4-6 containing arginine hydrochloride, as the solution pH decreased to 4.8, there was an increase in the formation of larger aggregates as well as an increase in dimeric species as the solution pH increased up to 5.4 (FIGURE 5B). At solution pH 5.1, formulation 6 with the presence of arginine hydrochloride had the lowest amount of total HMWS compared to formulation 2 without the presence of arginine hydrochloride. Furthermore, the behavior of formulation 7 demonstrated that an increase in acetate buffer concentration from 10 mM to 40 mM had a relatively smaller effect on HMWS formation.

REFERENCE EXAMPLE 4

This example demonstrates a relationship between pH and HMWS formation for different denosumab formulations comprising varying concentrations of denosumab.

Denosumab protein concentrations ranging from 15 mg/ml to 150 mg/ml were evaluated to assess the pH sensitivity of HMWS formation at various protein concentrations and at 75 mM arginine hydrochloride concentrations. Two pH values, i.e., pH 4.8 and 5.1, were evaluated at each of the tested protein concentrations: 15, 60, 120, and 150 mg/ml.

A total of 8 formulations (formulations 8-15; described in TABLE 4A) were evaluated in this study. To prepare these formulations, two aliquots of denosumab at 70 mg/mL in acetate at pH 5.2 were dialyzed against the respective DF buffer described in TABLE 4A. Both dialysis configurations #1 and #2 went through a total of 3 buffer changes to achieve a 1 million-fold dilution of the previous formulation to ensure complete buffer exchange. Following dialysis, aliquots were removed from each dialysis setup #1 and #2 described in TABLE 4A to prepare for the dilution step for formulations 8, 9, 12, and 13. The remaining material was then superconcentrated using a centrifuge-concentrator, followed by dilution to the corresponding denosumab concentrations listed in TABLE 4A and the addition of PS20 to a final concentration of 0.01%.

TABLE 4A

The formulations were packaged in containers with a fill volume of 1 mL and stored at 37°C for up to 1 month. Aggregation inhibition and stability against aggregation inhibition over time, based on the formation of HMWS and dimeric species, were evaluated using SE-UHPLC. The aggregation inhibition profiles of these formulations were compared under initial conditions and during and after storage.

FIGURE 6 represents a graph of the percentage of HMWS monitored by SE-UHPLC as a function of storage time at 37 °C for each formulation and TABLE 4B provides the data points for the graph.

TABLE 4B

Figures 7A and 7B show size exclusion chromatograms as a function of formulation after storage at 37°C for 1 month. As shown in Figure 6, the % HMWS increased as protein concentration increased. Formulations 8-11 at pH 4.8 had consistently higher levels of HMWS compared to the corresponding formulations at pH 5.1 (formulations 12-15). The increase in % HMWS at pH 4.8 is due to a large aggregation peak shown in Figure 7A (top) at approximately 5.75 minutes. Although the % HMWS at solution pH 5.1 had increasing dimeric species as protein concentration increased, the total HMWS was lower than the corresponding protein concentrations at solution pH 4.8 (Figure 7B (bottom)).

The difference in HMWS levels at pH 5.1 versus pH 4.8 became larger as denosumab concentration increased, with the difference being greater at higher denosumab concentrations.

EXAMPLE 5

Formulations with various concentrations of arginine, NAR, and two dipeptides consisting of arginine-arginine (Arg-Arg) and arginine-phenylalanine (Arg-Phe) were evaluated for stabilizing effects on solutions having a denosumab concentration of 120 mg/ml.

The formulations tested are described in TABLE 5 below. All acetate and excipient (except dipeptides) values quoted are for the buffer and excipient concentrations against which the antibody was diafiltered. Each dipeptide was added to the solution after buffer exchange at the level indicated in the table. Formulations R to X were achieved by UF/DF against the DF buffer listed below. Formulations Y and Z were achieved by LTF/DF, together in a single pool, against DF buffer containing 10 mM acetate, 3.6% sorbitol, pH 4.0. After LTF/DF, the pool of formulations Y and Z was split in 2, and then enriched for the Arg-Arg or Arg-Phe dipeptides from a 1 M stock solution containing 3.6% sorbitol at pH 5.1. Polysorbate 20 was added to each formulation to a final target concentration of 0.01%. Acetate is co-concentrated without arginine, resulting in a final acetate concentration of approximately 25 mM in formulations S through X. Sorbitol is preferentially excluded in the concentration process, resulting in a reduction of approximately 7 to 8% (w/v) from the initial concentration.

The formulations were packaged in containers at a fill volume of 1.0 ml. The formulations were stored at temperatures of 2 °C to 8 °C for up to 12 months and at 25 °C, 30 °C, and 37 °C for 3 months. Stability based on HMWS formation was evaluated using SE-UHPLC. The stability of these dipeptide formulations after one month at 37 °C was compared to arginine hydrochloride formulations at 37 °C as shown in Figure 8.

TABLE 5

Figure 8 shows the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37°C. The results show that amino acid aggregation inhibitors inhibited HMWS formation. The arginine-phenylalanine dipeptide, for example, showed significant improvement, resulting in approximately 0.3% less HMWS compared to the other formulations. The order of priority from lowest to highest HMWS was Z << V < Y = T = W = X = U < S < R. As can be seen in the figure, both the formulations containing the arginine-arginine (Arg-Arg) dipeptide (formulation Y) and arginine-phenylalanine (Arg-Phe) dipeptide (formulation Z) reduced HMWS formation compared to the control formulation lacking arginine and arginine-containing dipeptides (formulation R). Formulation Z contained the lowest amount of HMWS, higher than formulation Y

EXAMPLE 6

This example demonstrates the inhibition of denosumab aggregation and stability based on different concentrations of arginine and phenylalanine, and a comparative mixture of arginine and phenylalanine.

As described above, arginine hydrochloride (HCl) and arginine-phenylalanine HCl dipeptides were identified as reducing the initial starting level and rate of HMWS formation of denosumab. In this study, formulations containing arginine HCl concentrations, phenylalanine concentrations, and a combination of arginine HCl and phenylalanine were evaluated for stabilizing effects on solutions containing denosumab at 120 mg/mL.

The formulations tested (formulations 16-20) are described in TABLE 6A below. To prepare these formulations, an aliquot of denosumab at 70 mg/mL in acetate, pH 5.2, was dialyzed against the DF buffer described in TABLE 6A, with a total of 3 buffer changes to achieve a 1 million-fold dilution of the previous formulation to ensure complete buffer exchange. The material was then superconcentrated using a centrifuge-concentrator, followed by a dilution to 120 mg/mL and the addition of polysorbate 20 to a final concentration of 0.01%. Formulation 16 was considered the control formulation.

TABLE 6A

The formulations were packaged in containers with a fill volume of 1.0 mL. The formulations were stored at 37°C for up to 1 month. Aggregation inhibition and stability against aggregation inhibition over time, based on the formation of HMWS and dimeric species, were evaluated using SE-UHPLC. The aggregation inhibition profiles of these formulations were compared under initial conditions and during and after the storage period.

Figure 9 shows the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37°C. Figure 10 shows size exclusion chromatograms as a function of formulation after storage at 37°C for 1 month. Table 6B below shows the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37°C.

TABLE 6B

All formulations containing an amino acid aggregation inhibitor, arginine, or phenylalanine (formulations 17-20) were superior to the sorbitol control formulation lacking any amino acid aggregation inhibitor (formulation 16). All formulations containing phenylalanine (formulations 18, 19, and 20) contained similarly low levels of HMWS when compared to both the control and arginine HCl formulations (formulations 16 and 17, respectively) (Figure 9). The rate of HMWS formation was similar in the formulations containing arginine and phenylalanine HCl (formulations 17-19), as shown in Figure 9. The combination formulation comprising both 38 mM arginine and 38 mM phenylalanine (76 nM total, formulation 20) demonstrated better stability than the 75 mM arginine formulation (formulation 17) (Figure 9), but not better than the 75 mM phenylalanine formulation (formulation 19) (Figure 9).

EXAMPLE 7

This example demonstrates the inhibition of denosumab aggregation and stability as a function of different phenylalanine concentrations.

In previous studies, arginine hydrochloride and arginine hydrochloride-phenylalanine dipeptides were identified to minimize the initial starting level and rate of HMWS formation of denosumab. Formulations containing arginine hydrochloride, various concentrations of phenylalanine, and a combination of arginine hydrochloride and phenylalanine were evaluated for stabilizing effects on solutions containing denosumab at 120 mg/mL.

The formulations tested are described in Table 7A below. To prepare test samples A-E, an aliquot of denosumab at 70 mg/mL in acetate, pH 5.2, was dialyzed against the DF buffers described below, with a total of 3 buffer changes to achieve a 1 million-fold dilution of the previous formulation to ensure complete buffer exchange. The material was then co-concentrated to approximately 130 mg/mL to 150 mg/mL using a centrifuge-concentrator, followed by dilution to 120 mg/mL and the addition of polysorbate 20 to a final concentration of 0.01%. Formulation A was considered the control formulation.

The formulations were packaged in containers at a fill volume of 1.0 mL. The formulations were stored at 37 °C for up to 1 month. Stability based on HMWS formation was evaluated using SE-UHPLC. The stability profiles of these formulations were compared after one month at 37 °C with the sorbitol and arginine hydrochloride/sorbitol formulations at 37 °C as shown in Figure 11A.

To prepare test samples F-K, an aliquot of denosumab at 70 mg/mL in acetate, pH 5.2, was ultrafiltered/diafiltered (UF/DF) against the DF buffers described below for a total of 12 diavolumes to ensure complete buffer exchange. The material was then excess concentrated to approximately 200 mg/mL using ultrafiltration, followed by dilution to 120 mg/mL and the addition of polysorbate 20 to a final concentration of 0.01%. The acetate concentration was 20 mM in these formulations. Formulation F was considered the control formulation. All acetate and excipient values quoted are for the buffer and excipient concentrations against which the antibody was dialyzed.

The formulations were packaged in containers at a fill volume of 1.0 ml. The formulations were stored at 40 °C for up to 1 month. Stability based on HMWS formation was evaluated using SE-UHPLC. The stability profiles of these formulations were compared after one month at 40 °C with the sorbitol and arginine hydrochloride/sorbitol formulations at 40 °C as shown in Figure 11B.

TABLE 7A

Figure 11A and Table 7B show the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37°C. Figure 11B and Table 7C show the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 40°C. Figures 12A and 12B show size exclusion chromatograms as a function of formulation after storage at 37°C and 40°C for 1 month, respectively.

TABLE 7B

TABLE 7C

All phenylalanine formulations (formulations C, D, E, G, and K) contained lower levels of HMWS when compared to both the sorbitol and arginine hydrochloride/sorbitol formulations (formulations A and B, respectively). The arginine hydrochloride and phenylalanine combination formulation had similar stability when compared to the arginine/sorbitol formulation (formulation B). All formulations were superior to the sorbitol control formulation (formulations A and F).

EXAMPLE 8

This example demonstrates an evaluation of different amino acid aggregation inhibitors.

An evaluation of different amino acid aggregation inhibitors was performed by preparing eight formulations with a hydrophobic, aromatic, or polar/charged amino acid to determine their effect on minimizing the amount (%) of HMWS in a high-concentration (120 mg/mL) denosumab liquid formulation, and HMWS formation over time. The formulation included one of the eight L-amino acids and a reduced amount of sorbitol, relative to a control formulation that contained no amino acid aggregation inhibitors and a higher amount of sorbitol for isotonicity (formulation 26).

The amino acid aggregation inhibitors tested were grouped into one of three groups (groups I-III) and contained the amount of amino acid aggregation inhibitor as follows:

I. Aromatic amino acids:

(a) 38 mM Phenylalanine (formulation 27);

(b) Tryptophan 38 mM (formulation 28);

II. Polar/charged amino acids:

(a) 75 mM arginine HCl (formulation 29);

(b) 75 mM lysine (formulation 30);

(c) Histidine 75 mM (formulation 31);

III. Hydrophobic amino acids:

(a) Leucine 38 mM (formulation 32);

(b) 38 mM isoleucine (formulation 33);

(c) Valine 38 mM (formulation 34).

To prepare formulations 26-34, an aliquot of denosumab at 70 mg/mL in acetate, pH 5.2, was dialyzed against buffer DF described in Table 8A, with a total of 3 buffer changes to achieve a 1 million-fold dilution of the previous formulation to ensure complete buffer exchange. Dialysis of histidine formulation F used a buffer with an initial pH of 4.0, and the pH was predicted to shift to the target pH of 5.1 upon protein concentration due to the Donnan effect and acetate concentration. However, the pH did not shift to the target pH of 5.1 after the protein was concentrated to 120 mg/mL, but remained at pH 4.0. To bring the pH of the histidine formulation to pH 5.1, titration with dilute (0.1 N) NaOH was required. The remaining formulations were over-concentrated using centrifuge-concentrator units, followed by dilution to 124-128 mg/ml and the addition of polysorbate 20 to a final concentration of 0.01% (w/v).

TABLE 8A

The formulations were packaged in containers with a fill volume of 1.0 mL. The formulations were stored at 37°C for up to 4 weeks. Aggregation inhibition and stability against aggregation inhibition over time, based on the formation of HMWS and dimeric species, were evaluated using SE-UHPLC. The aggregation inhibition profiles of these formulations were compared under initial conditions and during and after the storage period.

Figures 13-15 represent graphs of the percentage of HMWS monitored by SE-UHPLC as a function of storage time at 37 °C for each formulation and Table 8B provides the data points for the graphs. Figures 16-18 show the chromatographic overlays of the formulations listed in Table 8A after storage at 37 °C for 1 month. Figures 13 and 16 refer to formulations comprising aromatic amino acids, Figures 14 and 17 refer to formulations comprising polar/charged amino acids, and Figures 15 and 18 refer to formulations comprising hydrophobic amino acids.

TABLE 8B

As shown in Figures 13-15, all formulations containing an amino acid aggregation inhibitor (formulations 27-34) demonstrated some improvement in stability relative to the acetate/sorbitol formulation (formulation 26). Formulations containing an aromatic amino acid (formulations 27 and 28) showed the greatest reduction in % HMWS. The formulation containing phenylalanine (formulation 27) also demonstrated a significant reduction in HMWS, and the formulation containing tryptophan showed the greatest reduction relative to the control (formulation 26). Of the denosumab formulations containing polar/charged amino acids (formulations 29-31), they generally showed higher amounts of higher-order aggregates (Figure 17) compared to the other formulations containing amino acid stabilizers (Figures 16 and 18), and this specific histidine formulation showed higher amounts of HWMS overall when compared to the acetate/sorbitol formulation (formulation 26) (Figure 14). The results for the histidine formulation could be skewed by the dialysis process, longer duration at pH 4.0, and titration of the formulation with dilute NaOH. All formulations containing a hydrophobic amino acid (formulations 32-34) demonstrated consistent enhancement in HWMS formation.

EXAMPLE 9

This example demonstrates a potential mechanism of action of arginine and phenylalanine in stabilizing denosumab. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a sensitive and robust technology for characterizing protein-protein/ligand/excipient interactions. The method detects changes in the hydrogen bonding of the amide backbone due to interaction with the excipient.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was performed on denosumab (at a concentration of 3 mg/ml) in 10 mM acetate buffer (pH 5.2) (“A52”) in the presence of L-arginine (formulation 35), L-phenylalanine (formulation 36), or L-glycine (formulation 37) and compared to a denosumab formulation lacking any amino acid aggregation inhibitors (formulation 38). Experiments were conducted at 4°C (with 75 mM L-arginine, L-phenylalanine, or L-glycine) and 37°C (with 150 mM L-arginine, L-phenylalanine, or L-glycine). After analyzing over 530 peptides, a small number of regions with significant conformational change were identified. Some representative peptides from these regions are captured in Figures 19-30.

Figures 19-24 are graphs of % deuterium incorporation at 4 °C as a function of time (log(s)) for light chain amino acids 28-33 (Figure 19), light chain amino acids 108-116 (Figure 20), light chain amino acids 125-132 (Figure 21), heavy chain amino acids 47-59 (Figure 22), heavy chain amino acids 243-253 (Figure 23), and heavy chain amino acids 392-399 (Figure 24) for each of formulations 35-38.

Figures 25-30 are graphs of % deuterium incorporation at 37 °C as a function of time (log (s)) for light chain amino acids 28-33 (Figure 25), light chain amino acids 108-117 (Figure 26), light chain amino acids 124-131 (Figure 27), heavy chain amino acids 47-59 (Figure 28), heavy chain amino acids 242-253 (Figure 29), and heavy chain amino acids 392-399 (Figure 30) for each of formulations 35-38.

These data support that Arg and Gly have a similar interaction effect on denosumab, although Arg had a slightly stronger HDX signature (conformational changes) on denosumab: strong stabilization at the Fab region LC 28-33; subtle stabilization at the Fab regions LC 108-132 and HC 47-59, Fc CH3 HC 392-399; and subtle destabilization at the Fc region CH2243-253. Without wishing to be bound by any particular theory, it is hypothesized that the effect of arginine hydrochloride is due to a combined preferential exclusion from the denosumab surface and weak surface interactions, whereas glycine functions by preferential exclusion.

However, phenylalanine did not show significant structural perturbation on denosumab. Without wishing to be bound by any particular theory, it is conceived that the stabilizing effect of phenylalanine could be through one or more of the following mechanisms: side chain interactions due to the lack of effect on the peptide backbone (no HDX signature); and/or cation-pi interaction with arginine/lysine side chains without affecting the backbone hydrogen bonding network.

EXAMPLE 10

This example demonstrates a possible mechanism of action of phenylalanine that stabilizes denosumab.

To study the specific effect of Phe on denosumab, a molecular dynamics simulation was performed. Specifically, the Fab domain of denosumab was solvated in a simulation box with excess Phe, and two 10-ns simulations were performed. Additionally, Phe residues bound to Fab for more than 90% of the time were selected for further analysis. Nine such occurrences were identified. In 5 of the 9 long-residence observations, the Phe residue was bound to the VH/VL (variable heavy/variable light) and CH/CL (constant heavy/constant light) regions. In one example, the Phe side chain was thought to be interacting with the side chains of hydrophobic residues (e.g., V93, Y95, and W112 of the heavy chain and A44 and P45 of the light chain) at the VH/VL interface. In another example, the side chain Phe ring was thought to be interacting with the NH3+ and COO(-) residue groups (e.g., T165 of the light chain and G171, V172, and T174 of the heavy chain) at the CH1 and CL interface. Without wishing to be bound by any particular theory, this observation led to the idea that the specific effect of Phe in mitigating denosumab aggregation is due to the interaction of the phenyl group with hydrophobic residues (e.g., R30, G31, R32, and Y33 of CDR1 of the light chain, A52 of CDR2 of the light chain, and M106 of CDR3 of the heavy chain) that form the heavy constant 1 (Hc) and light constant 1 (Lc) chain interface. This interaction is assumed to replace a previously hydrophobic surface with a relatively more charged (hence hydrophilic) surface of NH3(+) and COO(-) groups of the Phe excipient.

EXAMPLE 11

A stability assessment of multiple anti-RANKL antibody constructs (IgG1, IgG2, and IgG4 isotypes) was performed. As described above, both arginine HCl and phenylalanine minimized baseline HMWS and HWMS levels over time when compared to the acetate/sorbitol control formulation of denosumab (an IgG2 immunoglobulin). This assessment was performed by comparing the potential of Arg-HCl and Phe to reduce HMWS in formulations containing different anti-RANKL antibody constructs. The IgG1 and IgG4 constructs tested in this study contained the same complementarity determining regions (CDRs) when compared to denosumab but contained different constant domain scaffolds. The different IgG2 construct tested in this study had different CDRs relative to denosumab but contained the same constant domain scaffolds.

Each antibody construct tested was purified and concentrated from 8 mg/ml to 70 mg/ml using centrifugal concentration. Each concentrated volume was divided into three aliquots and then dialyzed against acetate buffer formulated with sorbitol, sorbitol/phenylalanine, and sorbitol/arginine hydrochloride to prepare formulations 39-47, as described in Table 9. Post-dialysis samples were over-concentrated to greater than 120 mg/ml using centrifugal concentration. The antibody protein was diluted to 120 mg/ml with the respective buffer.

TABLE 9

The formulations were packaged in glass vials with a fill volume of 1.0 mL. The formulations were stored at 37°C for up to 1 month. Aggregation inhibition and stability against aggregation inhibition over time, based on HMWS formation, were evaluated using SE-UHPLC. The aggregation inhibition profiles of these formulations were compared under initial conditions and after the storage period. The stability of these formulations after storage was compared within the immunoglobulin class.

Figures 31, 33, and 35 (and related Tables 10, 12, and 14 below) show the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37°C with immunoglobulins G (IgG1, IgG2, and IgG4, respectively). Figures 32, 34, and 36 (and related Tables 11, 13, and 15 below) show the percentage of low molecular weight species (LMWS, e.g., protein fragmentation) as monitored by SE-UHPLC as a function of formulation and time at 37°C with immunoglobulins G (IgG1, IgG2, and IgG4, respectively). Figures 37, 38, and 39 show overlays of size exclusion chromatograms as a function of formulation after storage at 37°C for t=4 wk.

TABLE 10: % HMW, IgG1 comparison (A, B, C) at 37°C for 4 weeks

TABLE 11: % LMWS, IgG1 comparison (A, B, C) at 37°C for 4 weeks

TABLE 12: % HMW, IgG2 comparison (D, E, F) at 37°C for 4 weeks

TABLE 13: % LMWS, IgG2 (D, E, F) compared at 37°C for 4 weeks

TABLE 14: % HMW, IgG4 comparison (G, H, I) at 37°C for 4 weeks

TABLE 15: % LMWS, IgG4 comparison (G, H, I) at 37°C for 4 weeks

As shown in Figures 31 and 32, the IgG1 molecule, which has a CDR region similar to the previous denosumab samples, showed an approximately 0.2% reduction in HMWS with the addition of phenylalanine compared to the acetate/sorbitol control formulation. The IgG2 samples, which have a different CDR and are represented in Figures 33 and 34, showed an increase in HMWS in the acetate/phenylalanine/sorbitol formulation compared to the acetate/sorbitol control formulation. The acetate/sorbitol and acetate/phenylalanine/sorbitol formulations had similar stability for the IgG4 sample type, with acetate/sorbitol/arginine having greater HMWS formation as shown in Figures 35 and 36. In all cases with the IgG1, IgG2, and IgG4 sample types, the acetate/sorbitol/arginine containing formulation showed increased HMWS degradation when compared to the acetate/sorbitol (control) and acetate/phenylalanine/sorbitol formulations.

Due to the large increase in protein fragmentation in the acetate/arginine/sorbitol formulation as depicted in Figures 37 and 38, the relationship between fragmentation and antibody isoform was shown in Figures 32, 34 and 36. It has been shown in the literature that fragmentation-mediated aggregation of monoclonal antibodies can result in antibodies stored at 37 °C [Perico N. et al., J. Pharm. Sci. (2009) 98, pgs.

[3031 - 3042]. This mechanism is possible in this evaluation since fragmentation is greatest in the acetate/arginine/sorbitol formulations. Fragmentation is minimized in the acetate/phenylalanine/sorbitol formulation, potentially leading to fewer HMWS species. The IgG4 sample type did not accelerate fragmentation or aggregation.

Based on the data collected in this study, as well as molecular modeling data from previous data collected with denosumab, a strong correlation can be established between the CDR amino acid sequence and the relative effect of reducing HMW with phenylalanine. A reduction in HMW species was observed in denosumab (IgG2) and the IgG1 variant that had identical CDR amino acids, but no reduction in HMW was observed in the IgG2 variant with different CDR domains. It would appear that the amino acid sequences contained within the CDR domains are susceptible to interaction with phenylalanine and subsequent inhibition of aggregation. The IgG4 molecule also had identical CDR regions when compared to denosumab, but minimal change in aggregation was detected over the course of the study. The IgG4 molecule differs from the IgG1 and IgG2 versions primarily by its hinge amino acid length and functionally active structure. Because IgG1 and IgG2 antibody isoforms have an extended structure commonly described as a "Y" shape, a CH1 domain of IgG4 Fab interacts with the CH2 domain to form a more compact structure [Aalberse R.C. et al., Immunology (2002), 105, pp. 9-19]. This compact structure might inhibit the fragmentation and aggregation reactions commonly observed with IgG1 and IgG2 forms.

EXAMPLE 12

A stability monitoring study is performed for formulated denosumab as described below and in Table 16 (formulations 51-55). Diafiltration buffers differ in acetate concentration and starting pH to produce final formulations with pH 5.1 at a concentration of 120 mg/ml denosumab. In addition, the sorbitol level is adjusted to maintain isotonicity of the final product (-300 mOsm/kg). Denosumab at 70 mg/ml is diafiltered against each buffer for over 7 volumes, then ultrafiltered to approximately 180 µg/ml and diluted with diafiltration buffer and polysorbate to the concentration of 120 mg/ml denosumab and 0.01% polysorbate 20. Stability is assessed using SE-UHPLC after storage at 37°C and shows that the stability of denosumab in these formulations is highly similar. Initial HMW species decrease slightly with increasing initial acetate concentrations. In contrast, aggregation rates improve slightly in formulations with lower acetate levels.

TABLE 16

REFERENCE EXAMPLE 13

The following example reports the results of studies on the effect of arginine on the stability of denosumab to chemical denaturation at three different pH values: 4.5, 4.8, and 5 (or 5.2).

All chemical denaturation experiments were carried out using an Unchained Labs HUNK instrument with a fluorescence detector. The excitation wavelength was 280 nm and emission scans were recorded between 300 and 500 nm. For each denaturation experiment, protein, buffer, and denaturant (guanidinium HCl) were dispensed into 36 wells with a linear increase in denaturant concentration, resulting in a 36-point curve for each condition. Curve-fitting software provided by the instrument manufacturer (Unchained Labs) was used to fit the data points. A two-state model was used since there was evidence of only a single transition (native ^ denatured). Experiments were carried out using 0-6 M urea in 10 mM acetate, 5.0% w/v sorbitol, and titrated to the required pH of 4.5, 4.8, or 5 (5.2). The denosumab protein concentration was 7 mg/ml in all experiments.

Figure 40 shows the isothermal chemical denaturation curves of denosumab in the absence of arginine, at pH 4.5, 4.8, and 5.0. In the absence of arginine, the C<1/2> of chemical denaturant required for 50% unfolding is similar under the three pH conditions tested.

Figure 41 shows the isothermal chemical denaturation curves of denosumab in the presence of 75 mM arginine HCl at pH 4.5, 4.8, and 5.2. There was a marked increase in chemical stability to denaturation at pH 5.2 when compared to pH 4.8 and 4.5. The C<1/2> increases in 1 M of the guanidinium HCl denaturant at pH 5.2 versus lower pH. Therefore, the protective nature of arginine is surprising and highly pH dependent.

EXAMPLE 14

The following example provides the results of studies on the effect of arginine and phenylalanine on the stability over time of high-concentration denosumab formulations in syringes.

In previous studies, arginine hydrochloride and phenylalanine were identified as reducing the initial baseline level and rate of HMWS formation of denosumab. In this study, formulations containing arginine hydrochloride, phenylalanine, and a combination of arginine hydrochloride and phenylalanine were evaluated for stabilizing effects in solutions containing 120 mg/mL denosumab and stored in syringes for up to three months at two different temperatures.

The formulations tested are described in Table 17 below. To prepare formulations 56-59, denosumab at 70 mg/mL in acetate, pH 5.2, was diafiltered against the diafiltration (DF) buffers described below for 8 volumes to ensure complete buffer exchange. The material was then ultrafiltered to greater than 180 mg/mL, followed by dilution to 120 mg/mL and the addition of polysorbate 20 to a final concentration of 0.01%. Formulation 56 was considered the control formulation. The acetate, arginine HCl, and phenylalanine values listed are for the DF buffer, and estimated levels in the final composition at 120 mg/mL denosumab are provided, taking into account the exclusion of excipient and the co-concentration of acetate when no other counterion is present. Viscosity was measured at 5 °C and 25 °C using a Paar modular compact rheometer at shear rates up to 1000 s-1 (inverse seconds). Formulations were packaged in prefilled glass syringes (PFS) at a fill volume of 1.0 ml. Parallel sets of syringes were stored at 25 °C for 3 months and 37 °C for 2 months, respectively. Stability was based on the formation of HMWS as assessed using SE-UHPLC.

TABLE 17

Figures 42 and 43 show the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 25°C for 3 months and 37°C for 2 months, respectively.

TABLES 18-21 show the same data in tabular form and also the increase in HMWS relative to initial HMWS levels.

TABLE 18: % HMWS level at 25°C for 12 weeks

TABLE 19: Increase in HMWS at 25°C over 12 weeks

TABLE 20: % HMWS level at 37°C for 8 weeks

TABLE 21: Increase in HMWS at 37°C over 8 weeks

This example shows that the addition of arginine, phenylalanine, and a combination of these each reduces the initial HMWS level (t=0) with high-concentration denosumab formulations. At 25°C, the increase in HMWS is reduced in the phenylalanine formulation 59 compared to the control formulation 56. At 37°C, formulations 57 and 59 have reduced HMWS formation compared to the sorbitol control formulation 56. The formulation containing both arginine HCl and phenylalanine formed HMWS at a higher rate at 37°C relative to the other formulations, indicating that the combination of these excipients is destabilizing for denosumab at these higher temperatures in this formulation.

Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" shall be deemed to imply the inclusion of a stated integer or stage or group of integers or stages, but not the exclusion of any other integer or stage or group of integers or stages.

Throughout the specification, where compositions are described as including components or materials, it is contemplated that the compositions may also consist essentially of, or consist of, any combination of the recited components or materials, unless otherwise described. Likewise, where methods are described as including particular steps, it is contemplated that the methods may also consist essentially of, or consist of, any combination of the recited steps, unless otherwise described. The disclosure described herein can be suitably practiced in the absence of any element or step not specifically disclosed herein.

Claims (14)

1. An aqueous pharmaceutical formulation comprising: - an anti-RANKL antibody at a concentration in the range of 100 to 140 mg/ml; and - L-phenylalanine or L-tryptophan 5 mM to 180 mM; wherein the aqueous pharmaceutical formulation has a pH in a range of 5.0 to 5.4; and wherein the anti-RANKL antibody comprises a heavy chain comprising SEQ ID NO: 14 and a light chain comprising SEQ ID NO: 13. 2. The aqueous pharmaceutical formulation of claim 1, wherein the anti-RANKL antibody is denosumab. 3. The aqueous pharmaceutical formulation of claim 1 or 2, wherein the concentration of the anti-RANKL antibody is 120 mg/ml ± 12 mg/ml. 4. The aqueous pharmaceutical formulation of any one of the preceding claims, comprising: (i) L-phenylalanine; or (ii) 20 mM to 50 mM L-phenylalanine or L-tryptophan, optionally 40 mM L-phenylalanine. 5. The aqueous pharmaceutical formulation of any one of the preceding claims, further comprising a tonicity modifier, optionally selected from the group consisting of: sorbitol, mannitol, sucrose, trehalose, glycerol and combinations thereof, optionally further comprising 1.0% (w/w) to 5.0% (w/w) of the tonicity modifier. 6. The aqueous pharmaceutical formulation of claim 5, wherein the tonicity modifier comprises sorbitol, optionally comprising 2.0% (w/w) to 5.0% (w/w) sorbitol, or 3.5% (w/w) to 5.0% (w/w) sorbitol, or 4.0% (w/w) to 5.0% (w/w) sorbitol. 7. The aqueous pharmaceutical formulation of any one of claims 1 to 4, which is free of sorbitol and/or free of any tonicity modifier. 8. The aqueous pharmaceutical formulation of any one of the preceding claims, further comprising a surfactant, optionally wherein the surfactant is selected from the group consisting of: polyoxyethylene sorbitan fatty acid esters, alkylaryl polyethers, poloxamers and combinations thereof, optionally further wherein the surfactant is polysorbate 20. 9. The aqueous pharmaceutical formulation of claim 8, comprising at least about 0.004% (w/v) surfactant, optionally comprising less than 0.15% (w/v) surfactant, optionally further comprising 0.005% (w/v) to 0.015% (w/v) surfactant. 10. The aqueous pharmaceutical formulation of any one of claims 1 to 9, comprising 5 mM to 60 mM buffer, optionally (i) comprising 5 mM to 50 mM, optionally 9 mM to 45 mM buffer, and/or (ii) wherein the buffer is acetate, citrate, succinate, phosphate or hydroxymethylaminomethane (Tris). 11. The aqueous pharmaceutical formulation of any one of claims 1 to 9, which is self-buffered. 12. The aqueous pharmaceutical formulation of any one of the preceding claims, having a pH of 5.0 to 5.2, optionally 5.1. 13. The aqueous pharmaceutical formulation of any one of the preceding claims: (a) having (i) a viscosity that is no more than 6 cP at 5 °C, (ii) a conductivity in a range of 500 pS/cm to 2000 pS/cm, (iii) an osmolality of 200 mOsm/kg to 500 mOsm/kg, (iv) less than 2% high molecular weight species (HMWS) and/or more than 98% of the major antibody peak, as measured by SE-UHPLC, after storage at 2 °C to 8 °C for at least 12 months; and/or (b) comprising less than 2% high molecular weight species (HMWS) and/or greater than 98% of the major antibody peak, as measured by SE-UHPLC, after storage at 2°C to 8°C for at least 36 months; and/or (c) comprising less than 2% high molecular weight species (HMWS) and/or greater than 98% of the major antibody peak, as measured by SE-UHPLC, after first storage at 2°C to 8°C for at least 36 months, followed by second storage at 20°C to 30°C for 1 month. 14. The aqueous pharmaceutical formulation of any one of the preceding claims, for use in (a) the treatment or prevention of a skeletal-related event (SRE) in a subject with bone metastases from solid tumors, (b) the treatment or prevention of an SRE in a subject who is a skeletally mature adult or adolescent with giant cell tumor of bone such that it is inoperable or where surgical resection is likely to result in intense morbidity, (c) the treatment of hypercalcemia of malignancy resistant to bisphosphonate therapy in a subject, (d) the treatment or prevention of an SRE in a subject with multiple myeloma or with bone metastases from a solid tumor, (e) the treatment of osteoporosis of postmenopausal women at high risk of fracture, (f) the treatment to increase bone mass in women at high risk of fracture receiving adjuvant aromatase inhibitor therapy for breast cancer, (g) the treatment to increase bone mass in men with high risk of fracture receiving androgen deprivation therapy for non-metastatic prostate cancer, (h) treatment to increase bone mass in men with osteoporosis at high risk of fracture, (i) calcium or vitamin D therapy, (j) treatment of giant cell tumor of bone in a patient in need thereof, (k) treatment of hypercalcemia of malignant tumor in a patient in need thereof, (l) treatment of osteoporosis in a patient in need thereof, or (m) increasing bone mass in a patient in need thereof.